Streptomyces species producing the streptovaricin complex

Morphological, cultural and physiological-biochemical properties ofStreptomyces sp. strain 1000 and its antibiotic production were investigated. Antibiotics 1011 (identical with the streptovaricin complex) and 1012 (with antibacterial action) were isolated from the cultural broth of this strain. The overproducing natural variant 1011 was isolated from the population of a strain producing antibiotic 1011 at a concentration of 1000 mg/L (activity of the parent strain represents 41 mg/L only). Comparative taxonomical characteristic ofStreptomyces sp. strain 1000 with strains fromS. spectabilis showed that the strain 1000 differed in some properties and antibiotic production being considered as a new variant ofS. spectabilis. The strain shows an expressed antibiotic activity against G+ as well as G− bacterial and yeasts.     

A New Toxic Substance,Teleocidin, Produced by Streptomyces

The production and isolation of a new toxic substance, Teleocidin, and its biological properties were previously reported^1,2). Thereafter it has been found that an other strain of Streptomyces produced such specific toxic substance as Teleocidin in its cultured mycellium. Comparative tests of these two purified crystalline powders showed the new toxic substance resembles Teleocidin closely though differs in certain chemical properties. Therefore, the original Teleocidin is designated Teleocidin A, whereas that produced by a new strain of Streptomyces is named Teleocidin B, which had been tentatively called as the SK-toxic substance.

From the results of the chemical studies of Teleocidin B and its hydrogenated derivative, which was easily obtained as a crystalline form by the catalytic hydrogenation of Teleocidin B with Adam’s catalyst, molecular formula, C₂₈H_39~41N₃O₂ was postulated for Teleocidin B.

It was also recognized that an alcoholic hydroxyl, a lactam ring and a heterocyclic ring like indole or pyrrole structure existed as the functional groups of Teleocidin B.     

Actinoplanic acids A and B as novel inhibitors of farnesyl-protein transferase

Actinoplanic acids A and B are macrocyclic polycarboxylic acids that are potent reversible inhibitors of farnesyl-protein transferase. Actinoplanic acids A and B were isolated from Actinoplanes sp. MA 7066 while actinoplanic acid B was isolated from both MA 7066 and Streptomyces sp. MA 7099. Actinoplanic acids A and B are competitive with respect to farnesyl diphosphate and are selective inhibitors of farnesyl-protein transferase because they do not inhibit geranylgeranyl-protein transferase type 1 or squalene synthase. MA 7066 is believed to be a novel species of actinomycetes while MA 7099 is believed to be a novel strain of Streptomyces violaceusniger on the basis of morphological, biochemical and chemotaxonomic characteristics as well as its production of actinoplanic acids.     

<Emphasis Type="Italic">Streptomyces fildesensis</Emphasis> sp. nov., a novel streptomycete isolated from Antarctic soil

A novel actinomycete strain, GW25-5^T, was isolated from a soil sample collected from the Fildes Peninsula, King George Island, West Antarctica. The strain was characterized by white to grey aerial mycelia, which were differentiated to straight to flexuous spore chains, with rod-shaped smooth spores. The cell wall of strain GW25-5^T contained LL-diaminopimelic acid (A₂pm) and traces of meso-A₂pm. Whole-cell sugars were galactose and minor amounts of mannose and glucose. The predominant menaquinones were MK-9(H₆) (49%), MK-9(H₈) (24%) and MK-9(H₄) (12%). The phospholipids contained DPG, PE, PI, PIM and PL(s). The major cellular fatty acids were iso-C_16:0 and anteiso-C_15:0. Genomic DNA G+C content of strain GW25-5^T was 70.0 mol%. BLAST result showed that strain GW25-5 has the 16S rRNA gene sequence highest similarity of 97.5% with members of genus Streptomyces and phylogenetic analysis indicated that this strain belongs to the genus Streptomyces. DNA–DNA relatedness values of strain GW25-5^T with the closest species of Streptomyces purpureus LMG 19368^T and Streptomyces beijiangensis YIM 6^T were significantly lower than 70% of the threshold value for the delineation of genomic species. A polyphasic taxonomic investigation based on a judicious combination of genotypic and phenotypic characteristics revealed that the organism represents a novel species of the genus Streptomyces. Thus, we propose strain GW25-5^T as the type strain of this novel species, Streptomyces fildesensis (=CGMCC 4.5735^T = YIM 93602^T = DSM 41987^T = NRRL B 24828^T).     

Cloning and characterization of a novel tyrosine ammonia lyase-encoding gene involved in bagremycins biosynthesis in Streptomyces sp.

Tyrosine ammonia lyase catalyzes the deamination of l-tyrosine to trans-coumaric acid. A novel tyrosine ammonia lyase-encoding gene, bagA, was cloned and sequenced from bagremycins-producing strain Streptomyces sp. Tü 4128 whose protein product contains a Ala–Ser–Gly segment in the active site. The disruption of the bagA gene abolished trans-coumaric acid and bagremycins production. trans-coumaric acid restored the formation of bagremycin A in the mutant, but not bagremycin B. Thus, trans-coumaric acid is a precursor for biosynthesis of bagremycins and the bagA gene codes for tyrosine ammonia lyase to synthesize trans-coumaric acid. This is a novel bacterial tal gene reported in actinomycetes for the second time and for the first time in a Streptomyces sp.     

Enhanced heterologous polyketide production in<Emphasis Type="Italic"> Streptomyces</Emphasis> by exploiting plasmid co-integration

A plasmid named pSMALL was discovered in a Streptomyces coelicolor strain that significantly enhanced the levels of production of 15-methyl-6-deoxyerythronolide B, a polyketide lactone normally produced in low amounts by engineered polyketide synthase (PKS) genes. It is a co-integrate between a conventional SCP2*-derived Streptomyces expression plasmid, pJRJ2, and SCP2@, a variant of the parental SCP2* plasmid. SCP2@ has a 45-bp deletion 35 bp upstream of the start codon of the repI gene in the replication region; and this correlated with an enhanced plasmid copy number and polyketide overproduction by its derivatives. This discovery was exploited to construct pBOOST, a high-copy-number cloning vector that can be used to achieve greatly elevated (at least 25-fold), stable metabolite production by PKS genes cloned in SCP2*-derived vectors by forming co-integrates with them.     

<Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="Italic">mayteni</Emphasis> sp. nov., a novel actinomycete isolated from a Chinese medicinal plant

An actinomycete strain, designated YIM 60475^T, was isolated from the roots of Maytenus austroyunnanensis and was characterized by using a polyphasic approach. The strain was determined to belong to the genus Streptomyces, based on its phenotypic and phylogenetic characteristics. The strain produced spiral spore chains on aerial mycelium. The cell wall contained ll-diaminopimelic acid. Whole-cell hydrolysates contained galactose, glucose, and xylose. The phospholipid was type II. The DNA G+C content of the type strain was 73.3 mol%. DNA–DNA hybridization and comparison of physiological and chemical characteristics suggested that strain YIM 60475^T is a new Streptomyces species, for which the name Streptomyces mayteni sp. nov. is proposed. The type strain is YIM 60475^T (=CCTCC AA 207005^T = KCTC 19383^T). Hua-Hong Chen and Sheng Qin contributed equally to this work.     

A lytic enzyme cocktail from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. B578 for the control of lactic and acetic acid bacteria in wine

Beside yeasts, lactic acid bacteria (LAB) are the most abundant microbes in must during vinification. Whereas Oenococcos oeni is commercially used as a starter culture for the biological acid reduction in wines, other species are responsible for different types of wine spoilage. Members of the genera Pediococcus, Weissella, Leuconostoc, and Lactobacillus are producers of exopolysaccharide slimes, biogenic amines, acetic acid, and other off-flavors. In order to control microbial growth, different procedures such as heating of must and addition of sulfite or lysozyme from egg white are generally applied. Yet, because of health risks, the application of sulfite should be reduced and lysozyme is not effective against all LAB. In this study, we describe exoenzymes from a Streptomyces sp. strain B578 lysing nearly all wine-relevant strains of LAB and Gram-negative acetic acid bacteria. The lytic enzymes were active under wine-making conditions, such as the presence of sulfite and ethanol, low temperatures, and low pH values. The analysis of the exoenzyme composition revealed a synergistic action of different cell wall hydrolases. In conclusion, the lytic cocktail of Streptomyces sp. B578 is a promising tool for the control of wine-spoiling bacteria. This article is dedicated to Prof. Dr. Ferdinand Radler on the occasion of his 80th birthday.     

Srinivasan’s coagulation test in taxonomically classified streptomycetes

Srinivasan’s coagulation test was performed in 18 strains of the genusStreptomyces and one strain of the genusActinoplanes. The highest coagulation activity was detected in strains systematically classified in a series of streptomycetes with pink or red aerial mycelium:S. erythreus, Streptomyces sp. AJ/22,S. roseo-luteus andS. griseofuscus. With the exception ofS. griseofuscus these three cultures also exhibited the highest inhibitory activity againstB. subtilis. When using hemoglobin as substrate it was possible to detect acid, neutral and alkaline proteinases with the highest proteolytic activity at pH 3.0 to 4.0 in the most active strain ofS. erythreus.     

Selective inhibition of the cyanobacterium, <Emphasis Type="Italic">Microcystis</Emphasis>, by a <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

A Streptomyces strain, NT0401, was isolated from soil that selectively inhibited Microcystis strains but did not affect other microorganisms. Based on its morphology, physiology and 16S rDNA sequence, it was identified as Streptomyces grisovariabilis. The active substance produced by NT0401 was a water-soluble compound with a Mr <1 kDa that was stable over a broad pH range and at 100°C for 20 min. This organism should be a potential environment-friendly strain for control of Microcystis blooms.     

Pyrindicin,a New Alkaloid from a Streptomyces Strain

In the course of our examination for the alkaloid productivities of Streptomyces strains, Streptomyces sp. NA–15 was found to produce a new alkaloid, pyrindicin, in the culture medium. The strain NA–15 was found to be a variant of Streptomyces griseoflavus and was designated as S. griseoflavus var. pyr indie us nov. var.

After the culture conditions for pyrindicin production were studied, pyrindicin was obtained as its hydrochloride (mp 145°C, decomp.) from the cultured broth. The compound was shown to possess weak antimicrobial and several pharmacological activities. The LD₅₀ of the hydrochloride (ip, in mice) was 87 mg/kg.     

Purification,Biochemical Characterization,and Cloning of Phospholipase D from <Emphasis Type="Italic">Streptomyces racemochromogenes</Emphasis> Strain 10-3

We previously isolated Streptomyces racemochromogenes strain 10-3, which produces a phospholipase D (PLD) with high transphosphatidylation activity. Here, we purified and cloned the PLD (PLD103) from the strain. PLD103 exerted the highest hydrolytic activity at a slightly alkaline pH, which is in contrast to the majority of known Streptomyces PLDs that have a slightly acidic optimum pH. PLD103 shares only 71–76% amino acid sequence identity with other Streptomyces PLDs that have a slightly acidic optimum pH; thus, the diversity in the primary structure might explain the discrepancy observed in the optimum pH. The purified PLD displayed high transphosphatidylation activity in the presence of glycerol, l-serine, and 2-aminoethanol hydrochloride with a conversion rate of 82–97% in a simple one-phase system, which was comparable to the rate of other Streptomyces PLDs in a complicated biphasic system.     

Optimized transformation of Streptomyces sp. ATCC 39366 producing leptomycin by electroporation

Streptomyces sp. ATCC 39366 produces leptomycin derivatives. Leptomycin B, a potent and specific inhibitor against the export of nuclear proteins, is the main product; however, the introduction of DNA into this strain is almost impossible, which has impeded its further use. We developed a Streptomyces sp. ATCC 39366 transformation protocol to introduce foreign DNA via electroporation. Various conditions were examined, including treatments of the cell wall with weakening agents, electroporation parameters, and DNA content. We found that only plasmid DNA isolated from a dam ^? ET12567 strain resulted in successful transformation. The mycelium growing in a yeast-peptone-dextrose medium supplemented with 1% glycine at 28°C on a rotary shaker (220 rpm) was more dispersed than those without supplementation and prone to electroporation. The maximum transformation efficiency of 8×10² CFU/μg plasmid DNA was obtained at a field strength of 13 kV/cm with a time constant of 13 ms (25-μF capacitor; parallel resistance, 600 Ω) using 1-mm electrocuvettes. The results of the transformations of two other Streptomyces species indicated that the optimized conditions established in this study might only be applicable to Streptomyces sp. ATCC 39366. However, this is the first report of successful transformation of Streptomyces sp. ATCC 39366, and will facilitate the construction of a gene knockout mutant in Streptomyces sp. ATCC 39366 to produce series of new leptomycin derivatives.     

Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2

Summary Five strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated se1uence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different. One of them contains neither pSAM1 not pSAM2. As this isolate produces spiramycin, these plasmids probably do not play an important role in spiramycin production. Apart from its intrinsic biological interest, pSAM2 may be useful in the construction of cloning vectors for S. ambofaciens. Very stable transformants might be obtained in certain strains of S. ambofaciens, because of the possibility of integration of the pSAM2 derivative vector.     

Streptomyces sp. Z-11-6, a novel producer of extracellular L-glutamate oxidase

Glutamate oxidase activity was studied in 1254Streptomyces strains isolated from the zonal soils of various regions of Russia and other countries. Seven strains proved to be producers of extracellular L-glutamate oxidase. The most active producer strain was identified, and the conditions of enzyme biosynthesis were optimized. A multistep mutagenesis-selection procedure allowed a genetically stable strain,Streptomyces sp. Z-11-6, to be obtained, whose glutamate oxidase activity was 40 times higher than that of the original natural isolate.     

Catalytic properties and stability of three common variants of placental alkaline phosphatase

Three placental alkaline phosphatases purified to homogeneity, i.e., the F, I, and S variants, were investigated for catalytic and stability properties. All three forms of the enzyme were found to have almost identical pH optima (10.7–10.8), similar sensitivity to the uncompetitive inhibitors L-phenylalanine (70%) and L-leucine (30%), and identical K_m values against p-nitrophenylphosphate, -glycerophosphate, and -naphthylphosphate. Significant differences among the three types were observed in thermal stability. The F variant was found to be most stable and the I variant most labile at 79 C. At 70 C all three forms were stable.This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 4217 and 03X-2725), from the Medical Faculty, University of Umeå, and Jubileumsklinikens i Umeå forskningsfond.     

Studio di una nuova specie di Streptomyces: Streptomyces nobilis sp. nov. e esame di ceppi affini appartenenti ai generi streptomyces,Streptoverticillium e Nocardia

Riassunto Alcuni ceppi di attinomiceti provenienti da diverso isolamento sono stati esaminati in comparazione con altri riferiti a specie o generi noti, onde procedere alla loro identificazione. I criteri diagnostici sono gli stessi di cui a precedenti nostre pubblicazione.I ceppi in studio sono riferibili a tre generi come da seguito: GenereStreptomyces. Sono riferiti aStreptomyces i ceppi 631 e 725, isolati da suolo, e sono descritti come specie nuovaStreptomyces nobilis sp. nov. Il ceppo 631 costituisce l'olotipo e il ceppo 725 una variante naturale.GenereStreptoverticillium. Viene riferito il ceppo in studio 174. Si è rinunziato ad una identificazione specifica limitando si all'inserimento nella serie Rubrireticuli della quale viene aggiornata la chiave delle specie.GenereNocardia. I ceppi 608 e 669 sono riferiti aNocardia asteroides e il ceppo 535 aProactinomyces (=Nocardia)ruber.

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Summary Strains of actinomycetes, isolated from different sources, have been examined comparatively together with cultures of known species and genera, in order to proceed to their identification. The diagnostic criteria followed are those described in previous papers.Our isolates have been referred to the following genera:GenusStreptomyces: to this genus belong our strains 631 and 725, isolated from soil and described as new species,Streptomyces nobilis sp. nov. Strain 631 is considered as the holotype and strain 725 as a natural variant.GenusStreptoverticillium: strain 174 belongs to this genus. No specific identification has been given, but the strain has been enclosed in the Rubrireticuli Series. A new key of the Series is provided.GenusNocardia: strains 608 and 669 are referred toNocardia asteroides, strain 535 toProactinomyces (=Nocardia)ruber.

     

Groundwater promotes emergence of asporogenic mutants of emetic Bacillus cereus

Bacillus cereus is a ubiquitous endospore-forming bacterium, which mainly affects humans as a food-borne pathogen. Bacillus cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR-F48, was isolated and characterized. VAR-F48 can survive in sterilized groundwater over a long period in a vegetative form and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR-F48 compared to its parental strain. The results revealed a significant re-routing of the metabolism in the absence of Spo0A. We concluded that VAR-F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A-mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments.     

<Emphasis Type="Italic">Streptomyces lividans</Emphasis> and <Emphasis Type="Italic">Brevibacterium lactofermentum</Emphasis> as heterologous hosts for the production of X<Subscript>22</Subscript> xylanase from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

The Aspergillus nidulans gene xlnA coding for the fungal xylanase X₂₂ has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum. Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii. B. lactofermentum was also able to produce xylanase X₂₂, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA. These production values are higher than those previously reported for the heterologous expression of the A. nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml). Moreover, the X₂₂ enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale.     

Histidine sensitive strain of the blue-green alga Nostoc muscorum: possible nif-his interactions

Summary A variant strain of the nitrogen-fixing bluegreen alga Nostoc muscorum, displaying impairment in its elemental nitrogen dependent growth and complete inhibition of growth by L-histidine in otherwise nitrogen-free medium, has been isolated and characterized for its response to L-glutamic acid and L-glutamine in presence of other inorganic nitrogen sources. A model based on the possibility of nif-his interaction has been proposed to account for the observed behaviour of the strain. It is inferred that the two sets of genes may occupy neighbouring positions on the blue-green algal genome.