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At least 19 recordsLinked to original sources

PubMed2026

Two Saccharopolyspora isolates from archaeological excavation sites: polyphasic taxonomy, biosynthetic potential, bioactivity profiling and description of Saccharopolyspora antiqui sp. nov.

Archaeological excavation sites represent underexplored microbial habitats with the potential to recover taxonomically and biotechnologically valuable actinomycetes. In this study, two Saccharopolyspora strains, 5N708T and 5N102, were isolated from soil samples collected from the Gaziantep-Doliche-Dülük and Bitlis-Ahlat-Selçuklu Cemetery archaeological excavation sites in Türkiye. A polyphasic taxonomic approach, including 16S rRNA gene sequencing, phylogenetic and phylogenomic analyses, average nucleotide identity, digital DNA-DNA hybridization, phenotypic characterization, and chemotaxonomic analyses, showed that strain 5N708T represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora antiqui sp. nov. is proposed, whereas strain 5N102 was assigned to Saccharopolyspora elongata. Both isolates were further evaluated for their antimicrobial, antioxidant, and cytotoxic activities, and their biosynthetic potential was investigated by genome mining. Both strains showed activity against Staphylococcus aureus, with strain 5N708T producing the larger inhibition zone. Strain 5N102 exhibited markedly stronger antioxidant activity than strain 5N708T in radical scavenging, ferric reducing antioxidant power, and reducing power assays. In contrast, strain 5N708T showed more promising cytotoxic activity, with relative selectivity toward MIA PaCa-2 pancreatic cancer cells compared with HEK293 cells after prolonged incubation. Genome mining revealed multiple biosynthetic gene clusters in both isolates, supporting their capacity to produce secondary metabolites. These findings indicate that archaeological soils are promising reservoirs of taxonomically novel and biologically active Saccharopolyspora strains.

Saccharopolyspora↗

PubMed2001

Saccharopolyspora flava sp. nov. and Saccharopolyspora thermophila sp.nov., novel actinomycetes from soil.

The generic position of two aerobic, Gram-positive, non-acid-alcohol-fast actinomycetes was established following the isolation of their PCR-amplified 16S rRNA genes and alignment of the resultant sequences with the corresponding sequences from representatives of the families Actinosynnemataceae and Pseudonocardiaceae. The assignment of the organisms to the genus Saccharopolyspora was strongly supported by chemotaxonomic and morphological data. The strains were distinguished both from one another and from representatives of validly described Saccharopolyspora species on the basis of a number of phenotypic properties. It is proposed that the organisms, strains 07T (= AS4.1520T = IFO 16345T = JCM 10665T) and 216T (= AS4.1511T = IFO 16346T = JCM 10664T), be classified in the genus Saccharopolyspora as Saccharopolyspora flava sp. nov. and Saccharopolyspora thermophila sp. nov., respectively.

Bacterial Typing Techniques↗

PubMed1988

Characterization of bacteriophage phi C69 of Saccharopolyspora erythraea and demonstration of heterologous actinophage propagation by transfection of Streptomyces and Saccharopolyspora.

A bacteriophage, designated phi C69, isolated from a culture of Saccharopolyspora erythraea was characterized. The phage propagates on Sac. erythraea NRRL 2338 but does not infect 10 Streptomyces or 3 Micromonospora species tested. It infects Sac. erythraea NRRL 2359 but does not produce infectious phage particles in this host. phi C69 is approximately 40 kb in length and contains cohesive ends. A cos fragment containing ligated phage DNA ends was cloned in Escherichia coli. Restriction maps of the phage DNA and the cos fragment for several enzymes are shown. Transfection of both Sac. erythraea and Streptomyces lividans with phi C69 resulted in approximately equal titres of infectious phage particles produced from approximately the same number of regenerating cells. Transfection of Sac. erythraea with DNA from Streptomyces phages SH10 and KC404 also resulted in the production of infectious phage particles. The basis for differences among hosts in susceptibility to infection by various actinophages is discussed.

Bacteriophages↗

PubMed1998

Saccharopolyspora spinosporotrichia sp. nov., a novel actinomycete from soil.

The generic position of an aerobic, Gram-positive, non-acid-alcohol-fast actinomycete was determined following isolation of the PCR-amplified 16S rRNA genes and alignment of the resultant sequence with corresponding sequences from representatives of the family Pseudonocardiaceae. The assignment of the organism to the genus Saccharopolyspora was strongly supported by chemotaxonomic and morphological data. The strain was distinguished from representatives of validly described Saccharopolyspora species by a number of phenotypic properties. It is proposed that the organism, strain AS4.198T, be classified in the genus Saccharopolyspora as Saccharopolyspora spinosporotrichia sp. nov.

Classification↗

PubMed1979

A new broad-spectrum aminoglycoside antibiotic complex, sporaricin. II. Taxonomic studies on the sporaricin producing strain Saccharopolyspora hirsuta subsp. Kobensis nov. subsp.

Morphological, cultural and physiological characteristics of a new nocardioform actinomycete are reported. The microorganism which produces the antibiotic complex sporaricin has been selectively isolated from a sample of soil obtained from Kobe City, Hyogo Prefecture, Japan. By whole-cell analysis of the actinomycete, meso-diaminopimelic acid, arabinose and galactose were identified. But lipid LCN-A (lipid characteristic of Nocardia) and nocardomycolic acid were not detected. The taxonomic characteristics of this strain is closely related to the genus of Saccharopolyspora, described by LACEY and GOODFELLOW. Based on the taxonomic comparison with Saccharopolyspora hirsuta ATCC 27875, the strain was considered to be a subspecies of Saccharopolyspora hirsuta. Therefore, the proposed subspecies is named Saccharopolyspora hirsuta subsp. kobensis.

Actinomycetales↗

PubMed1991

Biological characterization of induced phages from Saccharopolyspora hirsuta 367 and comparison with phage JHJ-1.

Phages JHJ-2 and JHJ-3 were isolated from Saccharopolyspora hirsuta 367 UC 8106 following induction with mitomycin C and amplified on S. hirsuta NRRL B-5792. Their properties were compared with those of phage JHJ-1, isolated previously from S. hirsuta 367 NRRL 12045. The DNA restriction patterns appeared to be identical. One-step growth experiments showed no differences between the replication cycles. Burst sizes ranged from 100 to 110 p.f.u. per cell. However, the three phages showed some differences in their behaviour in different hosts. The host range of phage JHJ-1, on non-lysogenic strains, was emended to include all of the Saccharopolyspora strains tested; the host range of phage JHJ-2 was shown to be identical to JHJ-1. Phage JHJ-3 did not form detectable plaques on strains of S. rectivirgula or S. erythraea except S. erythraea NRRL 2359. Neither phage JHJ-2 nor JHJ-3 formed plaques on any lysogenic strains, while JHJ-1 formed plaques on all such strains except S. hirsuta 367 UC8106. Phage JHJ-3 was characterized as a temperate bacteriophage because it formed turbid, self-limiting plaques and lysogenized S. hirsuta NRRL B-5792. It was spontaneously released from UC8106. Both JHJ-1 and JHJ-2 formed clear and invasive (Inv+ phenotype: the property to grow on old mycelium) plaques on some Saccharopolyspora strains but clear and self-limiting plaques on others. Thus, the expression of the Inv+ phenotype encoded by JHJ-1 and JHJ-2 appears to be modulated by the host cell.

Bacteriophages↗

PubMed2003

[Study on relationship between length of homologous sequences and chromosomic recombination rate in Saccharopolyspora erythraea].

In order to study the relationship between lengths of homologous fragments and chromsomic recombination rate in Saccharopolyspora erythraea, three homologous sequences, with mutant loci and different flanking sequences, (26bp + 27bp), (500bp + 576bp) and (1908bp + 1749bp), were synthesized by chemical reaction or PCR amplification, and cloned into pWHM3 to construct homologous recombination plasmids, pWHM1113, pWHM1116 and pWHM1119. When the plasmids were transformed into protoplast of Saccharopolyspora erythraea A226 under PEG mediated, on an average 30, 69 and 170 transformants grew on each plate for the three plasmids respectively, but chromosomic integration frequency were 0, 2% and 19% among corresponding transformants. Both pWHM1116 and pWHM1119 could take double crossover recombination, and exchange the mutant loci in the chromosome. It was concluded that when the flanking sequences were equal or more than (500bp + 576bp), they could take effective single and double recombination with Saccharopolyspora erythraea chromosome.

Chromosomes, Bacterial↗

PubMed1993

Estimation of the kinetic constants and elucidation of trends in growth and erythromycin production in batch and continuous cultures of Saccharopolyspora erythraea using curve-fitting techniques.

The kinetics of erythromycin production were dependent on the identity of the growth rate-limiting nutrient during batch cultures of Saccharopolyspora erythraea. Semilogarithmic linear regression provided a single estimate of growth rate during the exponential phase, but partial cubic spline curve fit derivatives provided time-dependent specific growth and production rate profile. Non-growth-linked product formation was observed when the medium was glucose- or phosphate-limited. However, growth-linked product formation was observed in a nitrate-limited medium. The kinetics observed in nitrate-limited chemostat culture provided evidence that Saccharopolyspora erythraea may be subject to noncompetitive inhibition by a growth-linked product under these conditions. A mathematical model was used to test this theory. The model simulation fitted the observed data very closely and was used to calculate estimates of the kinetic parameters involved: [formula; see text]

Biotechnology↗

PubMed1993

IS1136, an insertion element in the erythromycin gene cluster of Saccharopolyspora erythraea.

The Saccharopolyspora erythraea eryAI and eryAII genes, which, together with eryAIII, are responsible for the formation of the macrolactone portion of the antibiotic erythromycin, are separated by a 1.46-kb segment, designated IS1136, with the characteristics of an insertion sequence. It contains an open reading frame of 425 codons similar to that of the Anabaena IS891 and is present in four nonidentical copies in the Sac. erythraea genome. Inverted repeats were found near the ends of IS1136, and in the copy in eryA, one of the ends was found to overlap the 5' end of eryAII. Hybridization analysis suggests that IS1136 is confined to Saccharopolyspora species containing eryA-homologous DNA.

Amino Acid Sequence↗

PubMed1996

Structural studies of the major glycolipid from Saccharopolyspora genus.

A major glycolipid was isolated from the well characterized Saccharopolyspora species, S. hirsuta, S. rectivirgula, S. erythraea and one not completely identified strain (Saccharopolyspora sp.). On the basis of sugar and methylation analysis, specific enzymatic and chemical degradations of the carbohydrate moiety, its FAB mass spectrometry and NMR spectroscopy characterizations, the carbohydrate part was shown to be the glycerol linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->1/3)Gro. The internal mannose residue is esterified at C-6 by one fatty acid residue, whereas another fatty acyl chain substitutes the primary methylene position of glycerol. The main fatty acyl residues are anteiso-branched heptadecanoic acid and the iso-branched fatty acids iso-17:0, iso-16:0, and iso-18:0, with the former species being predominant. The major glycolipid has potential value for taxonomic and diagnostic purposes, especially in the specific diagnosis of farmer's lung disease.

Carbohydrate Conformation↗

PubMed2001

New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster.

Using a previously developed expression system based on the erythromycin-producing strain of Saccharopolyspora erythraea, O-methyltransferases from the spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14-membered polyketide macrolactone. The spnI, spnK and spnH methyltransferase genes were expressed individually in the S. erythraea mutant SGT2, which is blocked both in endogenous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryCIII. Exogenous 3-O-rhamnosyl-erythronolide B was efficiently converted into 3-O-(2'-O-methylrhamnosyl)-erythronolide B by the S. erythraea SGT2 (spnI) strain only. When 3-O-(2'-O-methylrhamnosyl)-erythronolide B was, in turn, fed to a culture of S. erythraea SGT2 (spnK), 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was identified in the culture supernatant, whereas S. erythraea SGT2 (spnH) was without effect. These results confirm the identity of the 2'- and 3'-O-methyltransferases, and the specific sequence in which they act, and they demonstrate that these methyltransferases may be used to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway. In contrast, 3-O-(2',3'-bis-O-methylrhamnosyl)-erythronolide B was found not to be a substrate for the 4'-O-methyltransferase SpnH. Although rhamnosylerythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosyl-erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl-erythronolide precursors using the S. erythraea strain SGT2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway. 3-O-(2'-O-methylrhamnosyl)-erythromycin D was tested and found to be significantly active against a strain of erythromycin-sensitive Bacillus subtilis.

Anti-Bacterial Agents↗

PubMed1999

New genus-specific primers for the PCR identification of members of the genera Pseudonocardia and Saccharopolyspora.

Members of the family Pseudonocardiaceae are difficult to identify on the basis of their micromorphology only. The biochemical characterization of each new isolate is a painstaking and time-consuming task which cannot always be undertaken when handling large numbers of strains as is the case in natural product screening programmes. In this study, two sets of genus-specific oligonucleotides were designed which allow rapid detection of members of the genera Pseudonocardia and Saccharopolyspora by means of PCR-specific amplification. The genus specificity of these primers was validated on a wide range of collection strains and the primers were subsequently used to study a group of 106 wild-type isolates that possessed morphological characteristics of the family. Out of this group, 51 strains could be identified as members of the genus Pseudonocardia and only nine isolates could be assigned to the genus Saccharopolyspora. The diversity indicated by whole-cell fatty acid profiles of both wild-type and reference strains was compared with that identified using the oligonucleotide primers. The partial 16S rDNA sequencing of representative wild-type strains was used to validate their genus assignment by PCR-specific amplification. This study shows the industrial usefulness of the application of these direct identification tools as well as the complementary use of two sources of data, PCR-specific amplification results and fatty acid composition, to assess the diversity of a microbial population.

Actinomycetales↗

PubMed2000

Studies on the interaction of fermentation and microfiltration operations: erythromycin recovery from Saccharopolyspora erythraea fermentation broths.

Changes in fermentation media not only affect the performance of the fermentation itself (with regard to the kinetics of biomass and product formation and the yields obtained) but also the initial product-recovery operations downstream of the fermentor. In this work, microfiltration experiments to remove Saccharopolyspora erythraea biomass from fermentation broth and to recover erythromycin were carried out using two fundamentally different media; a soluble complex medium (SCM) and an oil-based process medium (OBM). Small-scale batch fermentations of 14-L working volume were carried out in triplicate using both media. Broth samples were taken from each fermentation at regular intervals from the end of the exponential-growth phase onwards. These were then processed using a Minitan II (acrylic), tangential crossflow-filtration module, fitted with a single 60 cm(2) Durapore hydrophilic 0.2 microm membrane, operated in concentration mode. The OBM fermentations produced higher titers of erythromycin but required longer fermentation times due to increased lag phases and slower maximum-growth rates. The OBM also increased the loading on the membrane; at maximum product titers residual oil concentrations of 3 g. L(-1), antifoam concentrations of 2 g. L(-1) and flour concentrations estimated at approximately 10 g/L(-1) were typical. It was found that both the permeate flux and erythromycin transmission were affected by the choice of medium. The OBM had significantly lower values for both parameters (12.8 Lm(-2) h(-1) and 89.6% respectively) than the SCM (35.9 Lm(-2) h(-1) and 96.7% respectively) when the fermentations were harvested at maximum erythromycin titers. Transmission of erythromycin stayed approximately constant as a function of fermentation time for both media, however, for the OBM the permeate flux decreased with time which correlated with an increase in broth viscosity. The relatively poor microfiltration performance of the OBM medium was, however, offset by the higher titers of erythromycin that were achieved during the fermentation. The filtration characteristics of the SCM broth did not show any correlation with either broth viscosity or fermentation time. Image-analysis data suggested that there was a correlation between hyphal morphology (main hyphal length) and permeate flux (no such correlation was found for the OBM broth). Moreover, it has been shown for the OBM broth that the residual flour had a profound effect on the microfiltration characteristics. The influence of the residual flour was greater than that imposed by the morphology and concentration of the biomass. The understanding of the factors governing the interaction of the fermentation and microfiltration operations obtained in this work provides a first step towards optimization of the overall process sequence.

Biomass↗

PubMed2001

Viability, strength, and fragmentation of Saccharopolyspora erythraea in submerged fermentation.

Two fermentations of the commercially important erythromycin-producing filamentous bacterium Saccharopolyspora erythraea were conducted in defined media. One was glucose-limited and the other nitrate-limited. The viability of the hyphae was determined using the fluorescent stain BacLight (Molecular Probes, Eugene, OR). Also, the force required to strain hyphae to breakage was determined using micromanipulation and a sensitive force transducer. In both fermentations, fragmentation coincided with the appearance of regions in the mycelia with permeabilised membranes (considered nonviable). Under glucose-limitation, hyphal breaking force rose to 1,050 +/- 130 nN at the end of the growth phase and fell to an undetectable value as a result of glucose exhaustion. Under nitrate-limitation, hyphal breaking force fell from 900 +/- 160 nN during the growth phase to 550 +/- 40 nN in the stationary phase. In both cases image analysis showed that the dimensions of mycelia were of the same order, suggesting that the major factor influencing fragmentation was the appearance of nonviable regions (assumed to be weak). The location in which nonviable regions first appear within hyphae could not be determined because of their appearance coinciding with fragmentation.

Biomass↗

PubMed2001

Precursor-directed production of erythromycin analogs by Saccharopolyspora erythraea.

Diketide N-acetylcysteamine (diketide NAC) thioester precursors were fed to 6-Deoxyerythronolide B synthase (DEBS) ketosynthase-1 inactivated (KS1 degree) Saccharopolyspora erythraea strains to produce 13-substituted erythromycin analogs. This direct feeding process potentially represents a simplified production process over the current analog production system. Titers of these analogs were observed to increase linearly with the diketide concentration up to a precursor-specific saturation level. However, the rate of product formation was lower and the rate of diketide consumption higher with S. erythraea than was previously observed with a recombinant strain of Streptomyces coelicolor. Several strategies were pursued to address the issue of these high diketide consumption rates: (1) elucidation of the locale of diketide degradation, (2) addition of beta-oxidation inhibitors to the cultures, and (3) addition of a sacrificial diketide enantiomer to occupy putative degradative enzymes. Additionally, repeated addition of diketide to an S. erythraea KS1 degrees culture indicated that the titer of these erythromycin analogs is also currently limited by a shorter production period than observed during erythromycin synthesis by the parent strain. These results indicate potential avenues for expanding the use of this precursor-directed system from the generation of limited quantities of erythromycin analogs to a large-scale production system for these compounds.

Anti-Bacterial Agents↗

PubMed2002

Decreasing the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338 leads to increased resistance to breakage and increased antibiotic production.

Mutation and selection for increased resistance to cell-wall synthesis inhibitors led to alterations in the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338. Mutants with decreased branching frequency exhibited increased hyphal strength (estimated by in vitro micromanipulation). As the hyphal strength was increased, this led to a greater proportion of hyphal particles in liquid culture with a hyphal fragment diameter of greater than 88 microm. This, in turn, coincided with proportionately increased antibiotic production.

Bioreactors↗

PubMed2001

Strength of mid-logarithmic and stationary phase Saccharopolyspora erythraea hyphae during a batch fermentation in defined nitrate-limited medium.

A method for measuring mechanical properties of Saccharopolyspora erythraea is reported with data from a batch fermentation. Briefly, hyphae were glued to the end of a tungsten filament mounted horizontally on a sensitive force transducer. Free ends of hyphae were trapped against a flat surface by a second probe. The force transducer and tungsten filament were then moved at a fixed rate, the hypha were strained, and the force resisting motion recorded. From these data the maximum force resisting motion is taken as the force at which breakage occurs. Hyphae from the mid-logarithmic phase of a simple batch fermentation on defined medium were found to have a breaking force of 890 +/- 160 nN (95% confidence), while stationary phase hyphae were weaker at 580 +/- 150 nN. Video recordings of the experiments allowed an approximation of breaking strain, which did not differ significantly between samples at 0.18 +/- 0.03. Electron microscopy was used to measure cell wall thickness, cell diameter, and hence cell wall cross-sectional area. The ultimate tensile strength was estimated to be 24 +/- 3 MPa with no difference between the two samples, the lower breaking force of the stationary phase hyphae being attributed to a thinner cell wall. Assuming a linear relationship between stress and strain, the elastic modulus was estimated to be 140 +/- 30 MPa. These values are comparable with other structural biological materials such as yeast cell walls and collagen.

Anti-Bacterial Agents↗

PubMed2004

Identification of a phosphopantetheinyl transferase for erythromycin biosynthesis in Saccharopolyspora erythraea.

Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins (CPs) from fatty acid synthases (FASs) (primary metabolism), polyketide synthases (PKSs), and non-ribosomal polypeptide synthetases (NRPSs) (secondary metabolism). Bacteria typically harbor one PPTase specific for CPs of primary metabolism ("ACPS-type" PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism ("Sfp-type" PPTases). In order to identify the PPTase(s) associated with erythromycin biosynthesis in Saccharopolyspora erythraea, we have used the genome sequence of this organism to identify, clone, and express (in Escherichia coli) three candidate PPTases: an ACPS-type PPTase (S. erythraea ACPS) and two Sfp-type PPTases (a discrete enzyme (SePptII) and another that is integrated into a modular PKS subunit (SePptI)). In vitro analysis of these recombinant PPTases, with an acyl carrier protein-thioesterase (ACP-TE) didomain from the erythromycin PKS as substrate, revealed that only SePptII is active in phosphopantetheinyl transfer with this substrate. SePptII was also shown to provide complete modification of ACP-TE and of an entire multienzyme subunit from the erythromycin PKS in E. coli. The efficiency of the SePptII in phosphopantetheinyl transfer in E. coli makes it an attractive alternative to other Sfp-type PPTases for co-expression experiments with PKS proteins.

Acyl Carrier Protein↗