Rimosus Research Articles

Removal of toxic metals from aqueous mixtures. Part 1: Biosorption

The biosorption of toxic metals from an aqueous mixture containing zinc, copper and nickel, in the presence of calcium and sodium ions (usual co-existing cations in related systems) has been investigated. Industrial biomass samples of different origin have been examined batchwise as effective sorbents, including bacteria (Streptomyces rimosus), fungi (Penicillium chrysogenum) and yeasts (Saccharomyces carlsbergensis and Saccharomyces cerevisiae). The effect of solution pH was evaluated in the range of 3–11.5. Selectivity was observed, particularly for the removal of copper. The observed removal of metals by the application of biosorption was also compared in laboratory experiments with other more conventional separation techniques (filtration, centrifugation and flotation). © 1999 Society of Chemical Industry

Batch zinc biosorption by a bacterial nonliving Streptomyces rimosus biomass

The zinc biosorption capacity of a Streptomyces rimosus biomass was studied in the batch mode. After a heat pretreatment, optimum conditions of biosorption were found to be: an average saturation contact time of 4 h, a biomass particle size between 140 and 250 μm, the ambient temperature, a stirring speed of 250 rpm, and pH of 7.5. The equilibrium data could be fitted by a Langmuir isotherm equation. Under these optimal conditions, up to 30 mg Zn/g biomass, was fixed. Moreover, additional chemical treatment of the biomass by NaOH (1 mol/L), increased the biosorption capacity of about 80 mg Zn/g biomass.

Recombination between the linear plasmid pPZG101 and the linear chromosome of Streptomyces rimosus can lead to exchange of ends.

The 387kb linear plasmid pPZG101 of Streptomyces rimosus R6 can integrate into the chromosome or form a prime plasmid carrying the oxytetracycline biosynthesis cluster. The integration of plasmid pPZG101 into the linear chromosome of S. rimosus R6-501 in mutant MV25 was shown to be due to a single cross-over at a 4 bp common sequence. pPZG101 had integrated into a 250 kb DNA sequence that was reiterated at a low level. This sequence includes the oxytetracycline biosynthesis cluster, so that homologous recombination generated a mixed population carrying different copy numbers of the region. The 1 Mb linear plasmid pPZG103 in mutant MV17 had also arisen from a cross-over between pPZG101 and the chromosome, so that one end of pPZG103 consists of c. 850 kb of chromosomal sequence including the oxytetracycline biosynthesis cluster. The plasmid pPZG101 was shown to consist of a unique central region of about 30 kb flanked by terminal inverted repeats of about 180 kb. Analysis of a presumed ancestor plasmid pPZG102 suggested that the long terminal repeats had arisen by a recombination event during the strain development programme.

New genetic methods to improve secondary metabolite production in Streptomyces

The most potent chemical mutagens commonly used for yield enhancement in streptomycetes induce a limited spectrum of base-pair substitutions, heavily dominated by GC to AT transitions. The AT to CG transversion pathway complements the GC to AT pathway, but no strong mutagens with this specificity are available. However, mutT mutations in Escherichia coli enhance spontaneous AT to CG transversions about 1000-fold, so such mutations in streptomycetes could be very beneficial for random mutagenesis. Rate-limiting steps in secondary metabolite biosynthesis, on the other hand, can be best addressed by cloning and insertion of extra copies of the appropriate gene(s) into a neutral genomic site. This approach has been used successfully to improve the production of tylosin and pristinomycin. Transposon mutagenesis can be used to identify and clone neutral genomic sites, as demonstrated in Streptomyces fradiae to improve tylosin yields. Transposon mutagenesis can also be used to activate or enhance the transcription of genes important for secondary metabolite production, and to identify and clone both positive and negative regulatory elements. In some cases it may be advantageous to amplify the complete biosynthetic pathway for secondary metabolite production. In Streptomyces rimosus, a highly productive oxytetracycline producer was shown to contain the complete biosynthetic pathway near the end of the linear chromosome, and on a hybrid linear plasmid that had undergone recombination with the chromosome. This concept of linear replicon fusion might be applicable to other secondary metabolite pathway genes that are located near one end of the linear chromosome.

Novel metabolites of warfarin produced by Beauveria bassiana and Streptomyces rimosus : a novel application of hplc- nmr

1. Biotransformation of warfarin by the fungus Beauveria bassiana (ATCC 7159) yielded the first reported phase II warfarin metabolite, 3′,4′-dihydroxywarfarin-3′-[4- methoxyglucoside], another previously unreported metabolite, 3′, 4′-dihydroxywarfarin and also 4′-hydroxywarfarin. 2. Biotransformation of warfarin by Streptomyces rimosus (NRRL 2234) yielded the previously unreported metabolites, 12-hydroxywarfarin,4′-hydroxy-11-methoxywarfarin, and also 7-hydroxywarfarin and 4′-hydroxywarfarin, which have not been previously reported as biotransformation products from this organism. 3. Hplc-nmr has been used to identify biotransformation products of warfarin by S. rimosus directly from the microbial broth without prior isolation and purification.

Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker

The aminoglycoside 3′-phosphotransferase typeVIII ( APHVIII) encoding gene ( aphVIII) from Streptomyces rimosus was introduced by glass-bead high-efficiency transformation into the nuclear genome of green unicellular alga Chlamydomonas reinhardtii for induction of transformants resistant to aminoglycoside antibiotics. The aphVIII structural sequence was flanked by S. rimosus regulatory sequences which failed to direct expression in C. reinhardtii. The pSU937 plasmid containing these sequences was able to transform C. reinhardtii strain cw15 arg7–8 mt + for paromomycin resistance (Pm R) at a frequency (1.3–1.9) × 10 -7, probably as a result of in vivo gene fusion and expression of the aphVIII gene from regulatory elements of nuclear DNA. Evidence for the real C. reinhardtii transformation includes blot-hybridization with a probe specific for aphVIII and demonstration of APHVIII enzyme activity in crude cell extracts of transformants. Integrated Streptomyces DNA sequences, APHVIII enzyme activity and the aminoglycoside-resistance phenotype were stable through mitosis in the presence and absence of selection. The Pm R phenotype was inherited by the meiotic progeny of transformants from crosses with wild-type strains.

Oxidation and amidation of salicylate by Streptomyces species

Seven streptomycete strains were tested for biotransformation of salicylate. The products were identified by nuclear magnetic resonance spectroscopy and three types of conversion were found. Streptomyces cinnamonensis and Streptomyces spectabilis formed gentisate and salicylamide concurrently. Streptomyces rimosus transformed salicylate to salicylamide. Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus and Streptomyces avermitilis produced only gentisate. Time course studies of salicylate conversion by thin-layer chromatography and high pressure liquid chromatography showed that salicylamide was accumulated in the culture broth, whereas gentisate was further metabolized.Key words: salicylate, gentisate, salicylamide, biotransformation, Streptomyces spp.

Performance of industrial scale bioreactors with modified RUSHTON turbine agitators

AbstractThe data presented here with respect to the behaviour of industrial scale stirred tank bioreactors equipped with modified RUSHTON turbine agitators in the biosynthesis processes of antibiotics are valid for that case that the power consumption is the same as it is in standard RUSHTON turbine agitators.Each modified RUSHTON turbine agitator was obtained through the variation of the blade surface by adding perforations so that the ratio between the perforation surface area and the full surface area (or the surface fraction of the perforations) is 0.36.In the fermentations of Streptomyces aureofaciens, Streptomyces rimosus and Penicillium chrysogenum producing tetracycline, oxytetracyline and penicillin, respectively, in bioreactors equipped with modified RUSHTON turbine agitators, the relative antibiotic production is increased by more than 30% compared to standard bioreactors.

Microbial Demethylation of Immunosuppressant FK-506: Isolation of 31-O-FK-506-Specific Demethylase Showing Cytochrome P-450 Characteristics from Streptomyces rimosus MA187

As a result of an extensive screening program for the microbial modification of the immunosuppressant FK-506, one culture, Streptomyces rimosus MA187, which specifically catalyzed the C-31 demethylation of FK-506 was identified. Treatment of the biotransforming culture with FK-506 increased demethylase activity 2.4-fold and stabilized the cytochrome P-450 protein. The enzyme responsible for this demethylation (31-O-FK-506 demethylase) was isolated and shown to be a soluble cytoplasmic protein which is constitutively expressed in the cells, which requires NADPH, ferredoxin-NADP(sup+)-reductase, and ferredoxin for activity, and which shows a cytochrome P-450 light absorption characteristic. Carbon monoxide saturation of the enzyme preparation and known mammalian cytochrome P-450 inhibitors such as quinidine HCl, ketoconazole, troleandomycin, and sulfaphenazole abolish the demethylating activity extensively. The purified enzyme is a monomeric protein with a molecular mass of 42 kDa and shows its maximal activity at a pH of 7.4 and an incubation temperature of 34(deg)C. The first 19 N-terminal amino acids in the sequence of the purified protein have been determined, with no cytochrome P-450 match found in the OWL and Swiss-Prot 23 databases. The isolated demethylase is therefore a cytochrome P-450 protein that can be used as a catalyst for the synthesis of 31-O-desmethylFK-506, an important immunosuppressant and a known metabolite of FK-506 metabolism by human liver microsomes.

A Taxonomic Study of the Genus Streptomyces by Analysis of Ribosomal Protein AT-L30

The ribosomal AT-L30 proteins from 81 species of the genus Streptomyces as listed by Williams et al. in Bergey's Manual of Systematic Bacteriology were analyzed. My results provided further evidence that the genus Streptomyces is well circumscribed. On the basis of levels of AT-L30 N-terminal amino acid sequence homology, the strains were classified into four groups (groups I to IV) and a nongrouped category, whose members contained amino acid sequences characteristic of each species. A phylogenetic tree constructed on the basis of the levels of similarity of the amino acid sequences revealed the existence of six clusters within the genus. The first cluster contains the members of groups I and II together with several other species; the second cluster contains the members of groups III and IV and several other species; the third cluster contains Streptomyces ramulosus and Streptomyces ochraceiscleroticus; the fourth cluster contains only Streptomyces rimosus; the fifth cluster contains Streptomyces aurantiacus and Streptomyces tubercidicus; and the sixth cluster contains Streptomyces albus and Streptomyces sulphureus. Considerable agreement between the results of the AT-L30 analyses and the results of numerical phenetic classification was found, although there were numerous disagreements in details. For example, four groups (groups I to IV) defined by the AT-L30 analysis data did not correlate with the aggregate groups defined by numerical classification. In general, but not always, the species classified in a particular cluster in the numerical classification system had the same or similar AT-L30 terminal amino acid sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphogenesis, biomass and oxytetracycline production of Streptomyces rimosus in submerged cultivation.

The minimal concentration of adenosine triphosphate (ATP) which could be detected with spectrophotometry, HPLC and luciferin-luciferase methods was 1.0 microM, 3.3 microM and 100 nM, respectively. In submerged cultivation, most Streptomyces rimosus TM-55 was in hyphae fragment form at 65 h, became short-rod mycelia at 166 h, and lysed at 504 h incubation. The ATP content had maximal value at 24 h, then gradually decreased during cultivation. The oxytetracycline potency increased as incubation occurred, had maximal potency 178.9 micrograms/ml at 166 h, and then gradually decreased. Morphogenesis was very important in oxytetracycline production in submerged cultivation of Streptomyces; short-rod mycelia had high oxytetracycline production.

Rheological measurements of three Actinomycete in submerged cultures

Fermentation experiments were performed to obtain time-dependent data on broth rheology for three filamentous microorganisms, Streptomyces rimosus, Actinomadura roseorufa and Saccharopolyspora erythraea, cultivated under standard conditions in a mechanically stirred bioreactor. Rheological data were successfully analysed using Mitschka's technique and flow curves of the cultures described by the “power law” model in the range of shear rate between 5 and 100 s–1. Consistency coefficients of the cultures were found to vary continuously with fermentation time while flow behaviour index fell sharply within the first few hours of cultivation and then remained practically unchanged till the end of fermentation. The pH of the biomass was found to have a strong influence on both consistency coefficient and flow behaviour index.

Sequences of the oxytetracycline polyketide synthase-encoding otc genes from Streptomyces rimosus

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 β-ketoacyl synthase and acyl carrier protein found in other type-II (multi-component) PKS systems that specify construction of acetate-derived polyketide antibiotics.

Production of oxytetracycline by immobilized Streptomyces rimosus cells in calcium alginate gels

AbstractStreptomyces rimosus Pfizer 18234–2 cells were immobilized in calcium alginate and used for the production of oxytetracycline. The influence of the incubation period, alginate concentration and storage in CaCl2 were investigated. From the results of the repeated batch fermentations of the shake flasks, a good level of antibiotic was maintained for a period of about 28 days using 4% calcium alginate. The cell leakage and cell concentration inside the beads were affected by the alginate concentration and storage in CaCl2 solution.

Taxonomic Utility of Restriction Endonuclease Fingerprinting of Large DNA Fragments from Streptomyces Strains

Using a method known as low-frequency restriction fragment analysis (LFRFA) (M. L. Beyazova and M. P. Lechevalier, Int. J. Syst. Bacteriol. 42:422-433, 1992), we determined the molecular weights of Ase I restriction fragments of Streptomyces DNAs by pulsed-field gel electrophoresis. The levels of similarity of fragment patterns among strains were determined by using the simple matching coefficient, and clustering was performed by using the unweighted pair group with mathematical average algorithm. A total of 59 strains representing eight species and the numerically classified taxon Streptomyces cyaneus group A18 (S. T. Williams, M. Goodfellow, G. Alderson, E. M. H. Wellington, P. H. A. Sneath, and M. J. Sackin, J. Gen. Microbiol. 129:1743-1813, 1983) were studied. Forty-two strains (six species) formed eight clusters at levels of similarity of more than 80%; 17 strains (including the entire S. cyaneus group) were unclustered. Cluster 1 contained all of the Streptomyces albus strains studied plus two strains of Streptomyces somaliensis and two strains of Streptomyces lavendulae. Cluster 2 contained 8 of the 12 Streptomyces fradiae strains examined plus one strain each of S. somaliensis and S. lavendulae. Cluster 3 was heterogeneous in terms of species. Cluster 4 contained two S. somaliensis strains; cluster 5 contained three S. fradiae strains; cluster 6 contained three Streptomyces rimosus strains; and clusters 7 and 8 contained seven and three Streptomyces ipomoea strains, respectively. The S. cyaneus group strains exhibited no clustering among themselves or with the other species examined. Some Streptomyces species which exhibited high levels of similarity (85 to 95%) in physiological tests (e.g., S. albus and the S. fradiae strains in cluster 2) exhibited high levels of similarity in the LFRFA (84 and 81%, respectively). Other taxa (S. cyaneus group) which exhibited equally high levels of physiological similarity (90%) appeared to be unrelated as determined by the LFRFA. Species with lower levels of physiological similarity (e.g., S. somaliensis[75%], S. lavendulae[63%], and S. rimosus[68%]) exhibited low levels of LFRFA similarity (75, 64, and 54%, respectively). High levels of DNA-DNA relatedness (≫90%) (S. ipomoea) were reflected in high levels of similarity as determined by the LFRFA (75 to 100%); lower levels of DNA-DNA relatedness (ca. 70%) (S. cyaneus group) were reflected in low levels of LFRFA similarity (strains not clustered). We concluded that the presently used physiological tests reflect too small a portion of the genome to be universally useful in streptomycete species characterization. In contrast, high levels of DNA-DNA relatedness (≫90%) and high LFRFA similarity values will probably both be valuable in species delineation in actinomycetes.

Sequences of tRNA-encoding genes and associated open reading frames of Streptomyces lividans

A gene library of Streptomyces lividans has been screened for tRNA-encoding genes with labeled Streptomyces tRNA as a probe. By sequence analysis of hybridizing fragments, two single genes have been identified which code for tRNA Asp and tRNA Gly. Associated with the tRNA gly gene, there are three open reading frames (ORFs) which might code for gene products possibly involved in active transport processes through the bacterial membrane. The transcriptional organization of tRNA Gly and the following ORFs was examined by high-resolution S1 mapping. A third clone carried a cluster of genes which encode two tRNA Gln and three tRNA Glu. This cluster corresponds to a similar cluster previously described for Streptomyces rimosus [Plohl and Gamulin, Mol. Gen. Genet. 222 (1990) 129–134].

Use of date products in production of oxytetracycline by Streptomyces rimosus.

Use of date products in production of oxytetracycline by Streptomyces rimosus.

Transcriptional mapping of the promoter of the aminoglycoside acetyltransferase gene ( aacC9) of neomycin-producing Micromonospora chalcea

We have studied the promoter of the gene encoding aminoglycoside acetyltransferase ( aacC9) in neomycin-producing Micromonospora chalcea. S1 nuclease mapping showed that the transcription initiation point of this gene is at the translation start point, with no evidence of a conventional ribosome-binding site. The aac of paromycin-producing Streptomyces rimosus forma paromomycinus shows the same characteristics; there is no homology in the promoter regions of the two genes, whereas the coding sequences are very similar.

Characterization of an oxytetracycline‐resistance gene, otrA, of Streptomyces rimosus

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'.

Origin of tetracycline efflux proteins: conclusions from nucleotide sequence analysis

The sequences of six tetracycline efflux proteins and three transport proteins which have some resemblance to them were compared. The tetracycline efflux proteins fall into three families: (i) those encoded by pBR322, RP1, and Tn10 (Escherichia coli); (ii) pT181 (Staphylococcus aureus) and pTHT15 (Bacillus subtilis); and (iii) tet347 (Streptomyces rimosus). There is global sequence homology within each of the first two families, but there is none between the families. The pT181/pTHT15 family shares close homology with the N-terminal half of the methylenomycin A efflux protein (Streptomyces coelicor), while tet347 resembles the C-terminal half. Portions of the N-terminal half of the Tn10-encoded protein show significant resemblance to portions in the N-terminal half of the pT181/pTHT15 family, but this sometimes occurs among transport proteins which do not have a common substrate. Tetracycline efflux proteins, therefore, appear to have arisen on at least two, or possibly three, separate occasions, probably from other transport proteins.