Lapstatin, a new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits autogenous aminopeptidases

Lapstatin, a low-molecular-weight aminopeptidase inhibitor, was purified to homogeneity from Streptomyces rimosus culture filtrates. The purification procedure included extraction with methanol, followed by chromatography on Dowex 50WX4, AG50WX4, and HPLC RP C₁₈ columns. By amino acid analysis, mass spectrometry, and NMR spectroscopy, the structure of lapstatin was shown to be 3-amino-2-hydroxy-4-methylpentanoylvaline. Lapstatin inhibited the extracellular leucine aminopeptidases from Streptomyces rimosus, Streptomyces griseus, and Aeromonas proteolytica with an IC₅₀ in the range of 0.3–2.4 μM. IC₅₀ values for other enzymes tested were at least tenfold higher. Leucine aminopeptidase from Streptomyces griseus was inhibited in a competitive manner, with an inhibition constant of 5 × 10^–7 M. Lapstatin is the first low-molecular-weight compound isolated from streptomycetes shown to inhibit an autogenous aminopeptidase. Received: 7 December 1998 / Accepted: 29 March 1999     

Spore Germination and Mycelial Growth of Streptomycetes at Different Humidity Levels

This study is the first to show the ability of streptomycetes to develop at a very low humidity level. All of the streptomycetes studied produced growth at low humidity (a_w 0.86 and 0.67). This capacity was most markedly pronounced in Streptomyces odorifer, whose spores were capable of germinating, and mycelial germs increased in length, at the air humidity a_w 0.50. The formation of lateral branches (mycelium branching) at this humidity was noted only in single S. odorifer germs and only after 72 h of incubation. Study of streptomycete growth on an agarized medium with different osmotic pressures, created by various glycerol concentrations in the medium, showed that, at a_w 0.67, the spores of all the streptomycetes studied germinate, producing mycelial germs but not microcolonies. The ecological significance of mycelial prokaryotes in soil microbial communities that develop and function under conditions of extremely low humidity is discussed.     

Production of Hydrogen Sulfide by Streptomycetes and Methods for its Detection

The ability of streptomycetes to produce hydrogen sulfide is generally used for taxonomic purposes. It was found that the previously used method, the blackening of Peptone Iron Agar, does not clearly indicate formation of hydrogen sulfide. It was shown that the blackening of a lead acetate strip is the most accurate indicator for H₂S-producing streptomycetes. A great variety of organic and inorganic sulfur compounds were examined and compared, and the choice of the most suitable sulfur source and method for the detection of hydrogen sulfide is discussed.     

Glucose kinases from Streptomyces peucetius var. caesius

Glucose kinases (Glks) are enzymes of the glycolytic pathway involved in glucose phosphorylation. These enzymes can use various phosphoryl donors such as ATP, ADP, and polyphosphate. In several streptomycetes, ATP-glucose kinase (ATP-Glk) has been widely studied and regarded as the main glucose phosphorylating enzyme and is likely a regulatory protein in carbon catabolite repression. In cell extracts from the doxorubicin overproducing strain Streptomyces peucetius var. caesius, grown in glucose, a polyphosphate-dependent Glk (Pp-Glk) was detected by zymogram. Maximum activity was observed during the stationary growth phase (48 h) of cells grown in 100 mM glucose. No activity was detected when 20 mM glutamate was used as the only carbon source, supporting a role for glucose in inducing this enzyme. Contrary to wild-type strains of Streptomyces coelicolor, Streptomyces lividans, and Streptomyces thermocarboxydus K-155, S. peucetius var. caesius produced 1.8 times more Pp-Glk than ATP-Glk. In addition, this microorganism produced five and four times more Pp-Glk and anthracyclines, respectively, than its wild-type S. peucetius parent strain, supporting a role for this enzyme in antibiotic production in the overproducer strain. A cloned 726-bp DNA fragment from S. peucetius var. caesius encoded a putative Pp-Glk, with amino acid identities between 83 and 87 % to orthologous sequences from the above-cited streptomycetes. The cloned fragment showed the polyphosphate-binding sequences GXDIGGXXIK, TXGTGIGSA, and KEX₍₄₎SWXXWA. Sequences for the Zn-binding motif were not detected in this fragment, suggesting that Pp-Glk is not related to the Glk ROK family of proteins.     

Influence of transition metals on Streptomyces coelicolor and S. sioyaensis and generation of chromate-reducing mutants

Bacteria-assisted bioremediation is widely recognized as a low-cost method to minimize the consequences of soil pollution with toxic metals originating from industrial sites. Strains used in bioremediation have to deal with high metal load via biosorption, reduction, bioprecipitation, metal sequestration, and/or chelation. Actinobacteria, and streptomycetes in particular, are considered a perspective group for bioremediation as natural soil inhabitants with extensive secondary metabolism. Nevertheless, there is no reference information on survival of the model streptomycetes in the presence of the most abundant metal pollutants. Also, there are no reports describing the selection approaches towards improvement of bioremediation properties. In this work, the resistance of Streptomyces coelicolor M145 and Streptomyces sioyaensis Lv81 to certain transition metals and their growth under different pH values are described for the first time. Spontaneous chromate-resistant S. sioyaensis Lv81-138 strain was selected in the course of this work. Strain Lv81-138 is the most efficient actinobacterial Cr(VI) reducer reported so far, capable of converting 12 mmol/L of Cr(VI) into Cr(III) in a medium supplemented with 50 mmol/L K₂CrO₄.     

Identification of glucose kinase‐dependent and ‐independent pathways for carbon control of primary metabolism,development and antibiotic production in Streptomyces coelicolor by quantitative proteomics

Members of the soil‐dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient availability is a major determinant for the switch to development and antibiotic production in streptomycetes. Carbon catabolite repression (CCR), a main signalling pathway underlying this phenomenon, was so far considered fully dependent on the glycolytic enzyme glucose kinase (Glk). Here we provide evidence of a novel Glk‐independent pathway in Streptomyces coelicolor, using advanced proteomics that allowed the comparison of the expression of some 2000 proteins, including virtually all enzymes for central metabolism. While CCR and inducer exclusion of enzymes for primary and secondary metabolism and precursor supply for natural products is mostly mediated via Glk, enzymes for the urea cycle, as well as for biosynthesis of the γ‐butyrolactone Scb1 and the responsive cryptic polyketide Cpk are subject to Glk‐independent CCR. Deletion of glkA led to strong downregulation of biosynthetic proteins for prodigionins and calcium‐dependent antibiotic (CDA) in mannitol‐grown cultures. Repression of bldB, bldN, and its target bldM may explain the poor development of S. coelicolor on solid‐grown cultures containing glucose. A new model for carbon catabolite repression in streptomycetes is presented.     

云南酸性土壤中度嗜酸链霉菌的多样性

[目的]了解酸性土壤环境里中度嗜酸链霉菌的多样性,调查其物种资源.[方法]用分散和差速离心法及选择性分离培养基从14份云南酸性土壤样品中分离到367株具有链霉菌培养特征的放线菌,并进行了颜色分群.从各颜色类群中选取代表菌株共97株,通过显微形态观察和pH梯度生长实验确定其中的中度嗜酸链霉菌.进一步从中筛选出16株中度嗜酸链霉菌代表菌株,进行16SrRNA基因序列的相似性和系统发育分析,并结合基因组DNA-DNA相关性数据.[结果]分离菌株归为12同的颜色类群,其中80%属于中度嗜酸链霉菌,其代表菌株在系统发育树上形成了8个距离较远且与已知种不同的进化分枝,可能代表链霉菌属内至少8个不同的新基因种.[结论]用以上方法筛选出的中度嗜酸链霉菌可归为8个不同于已知种的进化群,说明云南酸性土壤含有丰富多样的中度嗜酸链霉菌新物种.     

Imaging secondary metabolism of Streptomyces sp. Mg1 during cellular lysis and colony degradation of competing Bacillus subtilis

Soil streptomycetes are saprotrophic bacteria that secrete numerous secondary metabolites and enzymes for extracellular functions. Many streptomycetes produce antibiotics thought to protect vegetative mycelia from competing organisms. Here we report that an organism isolated from soil, Streptomyces sp. Mg1, actively degrades colonies and causes cellular lysis of Bacillus subtilis when the organisms are cultured together. We predicted that the inhibition and degradation of B. subtilis colonies in this competition depends upon a combination of secreted factors, including small molecule metabolites and enzymes. To begin to unravel this complex competitive phenomenon, we use a MALDI imaging mass spectrometry strategy to map the positions of metabolites secreted by both organisms. In this report, we show that Streptomyces sp. Mg1 produces the macrolide antibiotic chalcomycin A, which contributes to inhibition of B. subtilis growth in combination with other, as yet unidentified factors. We suggest that efforts to understand competitive and cooperative interactions between bacterial species benefit from assays that pair living organisms and probe the complexity of metabolic exchanges between them.     

Unlocking Streptomyces spp. for Use as Sustainable Industrial Production Platforms by Morphological Engineering

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.     

Enzymes of ammonium assimilation inStreptomyces avermitilis

Glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine dehydrogenase (ADH) and alanine aminotransferase (GPT) were detected in the cell-free homogenate ofStreptomyces avermitilis grown in a defined medium containing ammonium sulfate as the only nitrogen source. At an initial NH₄ ⁺ concentration of 7.5 mmol/L, high activities of GS, GOGAT and GDH were found while that of ADH was low. The ADH activity was markedly increased at initially millimolar NH₄ ⁺ concentrations. In some characteristics of its NH₄ ⁺-assimilating system (e.g. control of some enzyme activities, the NADPH specificity of GOGAT, the presence of alanine aminotransferase),S. avermitilis differs from other known streptomycetes.     

A new streptomycete and a new polyene antibiotic,acmycin

A new antifungal antibiotic named acmycin was isolated from a soil streptomycete. Detailed comparative taxonomic studies showed that the organism differed from three related species of streptomycetes. The organism was referred to asStreptomyces sp. AC₂. The isolated antibiotic appears to be of polyene nature.     

Accumulation of Poly (3-Hydroxybutyric Acid) by Some Soil Streptomyces

In a limited-scale survey, 55 soil streptomycetes were screened for the accumulation of poly (3-hydroxybutyrate) [PHB]. Only 18% of the isolates accumulated PHB ranging between 1.9–7.8% of the dry biomass. The promising isolate DBCC-719, identified as Streptomyces griseorubiginosus, accumulated PHB amounting to 9.5% of the mycelial dry mass in the early stationary phase when grown in chemically defined medium with 2% (wt/vol) glucose as the sole source of carbon. Nitrogen-limiting conditions were inhibitory to growth and PHB accumulation. The isolated polymer was highly soluble in chloroform, gave a sharp peak at 235 nm on digestion with concentrated H₂SO₄, and had a characteristic infrared spectrum. Received: 26 March 1999 / Accepted: 3 May 1999     

Biochemical characterization of resistance determinants cloned from antibiotic-producing streptomycetes.

Determinants of antibiotic resistance have been cloned from four antibiotic-producing streptomycetes into Streptomyces lividans. Biochemical analyses of resistant clones revealed the presence of enzymes that had previously been characterized as likely resistance determinants in the producing organisms. These included: 23S rRNA methylases from S. azureus and S. erythreus, which confer resistance to thiostrepton and erythromycin, respectively; viomycin phosphotransferase from S. vinaceus; and aminoglycoside phosphotransferase and acetyltransferase from the neomycin producer S. fradiae. In general, the levels of antibiotic resistance of the clones were similar to those of the producing organisms. Although the two aminoglycoside-modifying enzymes from S. fradiae could independently confer only low-level resistance to neomycin, the presence of both enzymes in the same strain resulted in a level of resistance comparable with that of the producing organism.     

Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.     

Atmospheric Hydrogen Scavenging: from Enzymes to Ecosystems

We have known for 40 years that soils can consume the trace amounts of molecular hydrogen (H₂) found in the Earth''s atmosphere. This process is predicted to be the most significant term in the global hydrogen cycle. However, the organisms and enzymes responsible for this process were only recently identified. Pure culture experiments demonstrated that several species of Actinobacteria, including streptomycetes and mycobacteria, can couple the oxidation of atmospheric H₂ to the reduction of ambient O₂. A combination of genetic, biochemical, and phenotypic studies suggest that these organisms primarily use this fuel source to sustain electron input into the respiratory chain during energy starvation. This process is mediated by a specialized enzyme, the group 5 [NiFe]-hydrogenase, which is unusual for its high affinity, oxygen insensitivity, and thermostability. Atmospheric hydrogen scavenging is a particularly dependable mode of energy generation, given both the ubiquity of the substrate and the stress tolerance of its catalyst. This minireview summarizes the recent progress in understanding how and why certain organisms scavenge atmospheric H₂. In addition, it provides insight into the wider significance of hydrogen scavenging in global H₂ cycling and soil microbial ecology.     

Streptomyceten als Bildner industriell und diagnostisch bedeutsamer Enzyme

This review deals with known natural enzyme activities of streptomycetes and their applications. This knowledge may help potential users to exploit these organisms for individual purposes. The data given in tables are based on scientific publications and the IUPAC recommendations regarding nomenclature are followed. Included are only enzymes already utilized, with those of the primary metabolism being neglected.     

High-yield production of coenzyme F420 in Escherichia coli by fluorescence-based screening of multi-dimensional gene expression space

Coenzyme F₄₂₀ is involved in bioprocesses such as biosynthesis of antibiotics by streptomycetes, prodrug activation in Mycobacterium tuberculosis, and methanogenesis in archaea. F₄₂₀-dependent enzymes also attract interest as biocatalysts in organic chemistry. However, as only low F₄₂₀ levels are produced in microorganisms, F₄₂₀ availability is a serious bottleneck for research and application. Recent advances in our understanding of the F₄₂₀ biosynthesis enabled heterologous overproduction of F₄₂₀ in Escherichia coli, but the yields remained moderate. To address this issue, we rationally designed a synthetic operon for F₄₂₀ biosynthesis in E. coli. However, it still led to the production of low amounts of F₄₂₀ and undesired side-products. In order to strongly improve yield and purity, a screening approach was chosen to interrogate the gene expression-space of a combinatorial library based on diversified promotors and ribosome binding sites. The whole pathway was encoded by a two-operon construct. The first module (“core”) addressed parts of the riboflavin biosynthesis pathway and F_O synthase for the conversion of GTP to the stable F₄₂₀ intermediate F_O. The enzymes of the second module (“decoration”) were chosen to turn F_O into F₄₂₀. The final construct included variations of T7 promoter strengths and ribosome binding site activity to vary the expression ratio for the eight genes involved in the pathway. Fluorescence-activated cell sorting was used to isolate clones of this library displaying strong F₄₂₀-derived fluorescence. This approach yielded the highest titer of coenzyme F₄₂₀ produced in the widely used organism E. coli so far. Production in standard LB medium offers a highly effective and simple production process that will facilitate basic research into unexplored F₄₂₀-dependent bioprocesses as well as applications of F₄₂₀-dependent enzymes in biocatalysis.     

Extracellular metalloendopeptidase of Streptomyces rimosus

Metalloendopeptidase was isolated from Streptomyces rimosus culture filtrates in a homogeneous form. It was determined to be a 15 kDa basic protein, most active around pH 7.5, and susceptible to inhibition by chelating agents, N-bromosuccinimide, thiorphan, and 10⁻⁴ M zinc. The enzyme was highly specific for phenylalanine at the N-side of endopeptide bonds. Determination of amino acid sequence of the enzyme’s NH₂-part allowed the recognition of its structure homology with isolated and predicted metallopeptidases from several Streptomyces species. The data contribute to the definition of M7 family of metalloendopeptidases in streptomycetes.     

Use of electrophoretic enzyme patterns for streptomycete systematics

Abstract The relative electrophoretic mobilities of various enzymes from 24 different streptomycetes were determined on polyacrylamide gels in order to examine the relatedness of species and strains of the genus Streptomyces . Of 11 different enzymes tested in this study, hexokinase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase showed a limited number of constant and reproducible polymorphic enzyme patterns, by comparing which the inter-specific relationships could be examined. In contrast, glucose dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyrate dehydrogenase, phosphoglucose isomerase, peroxidase and esterase exhibited either weak non-reproducible or highly heterogeneous band patterns which were suitable for dissecting the strains within a species and a cluster group.