Screening and Identification of the Phospholipase D-producing Streptomyces

About 2,000 strains of streptomycetes and 200 strains of molds were tested for egg yolk-degrading activity. A strain isolated from soil was found to produce the strong activity in the culture medium.

The egg yolk-degrading substance was found to be phospholipase D and the producing strain was identified as Streptomyces hachijoensis. It was found out that the strains producing egg yolk-degrading substance appeared with high frequency in whirl-forming species in genus Streptomyces.     

Classification of novel soil streptomycetes as Streptomyces aureus sp. nov., Streptomyces laceyi sp. nov. and Streptomyces sanglieri sp. nov

The taxonomic positions of soil isolates known as Streptomyces groups A, B and C were clarified. Comparative 16S rDNA sequence studies indicated that representatives of all three taxa formed distinct phyletic lines within the Streptomyces tree though the group A strains were shown to be related to Streptomyces griseus and associated validly described species. The taxonomic integrity of all three groups was highlighted by DNA:DNA relatedness and ribotype data though the group A strains encompassed a higher degree of genetic variation than the group B and C strains. In light of these and earlier phenotypic data it is proposed that Streptomyces groups A, B and C be given species status as Streptomyces sanglieri sp. nov., Streptomyces aureus sp. nov. and Streptomyces laceyi sp. nov., respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Changes of Lysine- and Ornithine-lipids in Streptomyces sioyaensis

Lysine-lipid (siolipin A) and ornithine-lipid (siolipin B) were found at the same time in Streptomyces sioyaensis. They were also found in mutant strains (Lys^?, Met^?, Try^?, His^?) of Streptomyces sioyaensis. Ratio of siolipin A to siolipin B differed, depending on the culture conditions. The young mycelium contained siolipin A predominantly, while the aged mycelium did much more siolipin B. They also varied according to pH of the broth. As the whole, the effect was more conspicuous in the mycelium from jar fermenter than that from Sakaguchi flasks.     

Antimycin A-producing nonphytopathogenic Streptomyces turgidiscabies from potato

Aim:  To detect if substances with mammalian cell toxicity are produced by Streptomyces turgidiscabies and Streptomyces scabiei isolated from potato scab lesions. Methods and Results:  In vitro cultures of phytopathogenic and nonphytopathogenic strains of S. scabiei and S. turgidiscabies, isolated from scab lesions of potato tubers originating from nine different cultivars from Finland and Sweden, were tested for toxicity using the rapid spermatozoan motility inhibition assay, previously shown useful in the detection of many different Streptomyces toxins and antimicrobial compounds. Purified toxins were used as reference. Three nonphytopathogenic strains of S. turgidiscabies were found to produce antimycin A when cultured on solid medium. Conclusions:  Boar sperm-motility-inhibiting substances are produced by strains of S. turgidiscabies and S. scabiei. The most powerful inhibitory substance, produced by three nonphytopathogenic S. turgidiscabies strains, was identified as antimycin A. The phytotoxic compounds thaxtomin A and concanamycin A did not inhibit sperm motility even at high doses. Significance and Impact of the Study:  The presence of antimycin A-producing Streptomyces strains, nonpathogenic to potato, was unexpected but important, considering the high mammalian toxicity of this cytochrome bc-blocking antibiotic.     

Genetic analysis of the φC31 -specific phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2)

The phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) was shown to be specific to φC31 homo-immune phages, and to be absent from the closely related strain Streptomyces Iividans. A 16 kb fragment of S. coelicolor A3(2) DNA was isolated which complemented the Pgl^? phenotype of J1501, a pgl mutant derivative of the Pgl^tsS. coelicolor strain M130. The cloned DNA complemented only half of the available pgl mutants, which therefore represented at least two groups, designated Pgl class A and class B strains. It follows that more than one kind of high-frequency genetic event can lead to the Pgl^? phenotype. Crosses between class A and class B strains yielded high frequencies of Pgl⁺ recombinants. Crosses between strains of the same class gave no Pgl⁺ recombinants. The cloned DNA was altered by deletion or apparent point mutation upon passage through the two class B strains tested, such that it was no longer capable of complementing class A strains. This accumulation of mutations might suggest that the expression of the cloned DNA is toxic to at least some class B strains. The nature of the genetic instability associated with the Pgl system was not detectable by Southern blot analysis.     

Production of L-leucine aminopeptidase by selected Streptomyces isolates

Aims: To screen various Streptomyces cultures producing l ‐leucine aminopeptidase (LAP). Methods and Results: Twenty‐one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B‐3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8·0–8·5 and the temperature optima varied between 50 and 65°C. LAP of S. mobaraensis was stable at 60°C and pH 8·5 for 60 min. Metal ion salts, CoCl₂.6H₂O and ZnSO₄.7H₂O in 0·7 mmol l^?1 concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. Conclusions: Streptomyces mobaraensis NRRL B‐3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. Significance and Impact of the Study: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N‐terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis

Ivermectin, 22, 23-dihydroavermectin B1, is commercially important in human, veterinary medicine, and pesticides. It is currently synthesized by chemical reduction of the double bond between C22 and C23 of avermectins B1, which are a mixture of B1a (>80%) and B1b (<20%) produced by fermentation of Streptomyces avermitilis. The cost of ivermectin is much higher than that of avermectins B1 owing to the necessity of region-specific hydrogenation at C22–C23 of avermectins B1 with rhodium chloride as the catalyst for producing ivermectin. Here we report that ivermectin can be produced directly by fermentation of recombinant strains constructed through targeted genetic engineering of the avermectin polyketide synthase (PKS) in S. avermitilis Olm73-12, which produces only avermectins B and not avermectins A and oligomycin. The DNA region encoding the dehydratase (DH) and ketoreductase (KR) domains of module 2 from the avermectin PKS in S. avermitilis Olm73-12 was replaced by the DNA fragment encoding the DH, enoylreductase, and KR domains from module 4 of the pikromycin PKS of Streptomyces venezuelae ATCC 15439 using a gene replacement vector pXL211. Twenty-seven of mutants were found to produce a small amount of 22, 23-dihydroavermectin B1a and avermectin B1a and B2a by high performance liquid chromatography and liquid chromatography mass spectrometry analysis. This study might provide a route to the low-cost production of ivermectin by fermentation.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was made to characterize the active substance for the extraordinarily strong adjuvant effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) type 1 Kasuya strain. CPS-K was fractionated into acidic and neutral CPS-K by the addition of cetyl-pyridinium chloride. Neutral CPS-K exhibited an extremely strong adjuvant effect. The active substance in neutral CPS-K was precipitable when mixed with a rabbit homologous antiserum. The neutral CPS-K antigen was serologically distinct from the O antigen and from the acidic CPS-K which was the type-specific capsular antigen. Among preparations of neutral CPS-K from eight different strains of K. pneumoniae tested, the preparation from only one strain (MH-2) exhibited a strong adjuvant effect comparable to that of the neutral CPS-K from the Kasuya strain. The neutral CPS-Ks from Kasuya and MH-2 strains were antigenically identical. This antigen was not found in all preparations of neutral CPS-Ks obtained from seven different strains. Preparations of acidic CPS-Ks from all strains of K. pneumoniae tested with various serologic types including Kasuya and MH-2 strains were found to exhibit only weak adjuvant effects. The active substance (neutral CPS-K antigen from Kasuya strain) was shown to form a single peak upon analyses by gel filtration (Sephadex G-100) and ultracentrifugation. Sedimentation coefficient of the substance was approximately 20 S at a concentration of 5 mg per ml in 0.1 M NaCl. The active substance finally purified by gel filtration contained 65% sugars (as glucose equivalents), 6.8% hexuronic acids, 2.6% hexosamine, 2.3% proteins, and very small amounts of lipids.     

Microcycle sporogenesis in some streptomycetes without shift down treatment

Conditions for microcycle sporogenesis in two streptomycete strains without shifting the culture were found. The sporulation in Streptomyces granaticolor took place after 24 h of cultivation. The dry mass was increasing till 32 h probably due to production of a hydrophobic substance resembling fibrous sheath of aerial hyphae and spores. Ultrathin section of microcycle spores are presented.     

Studies on the Utilization of Hydrocarbons by Microorganisms

During the course of an investigation of the microbial assimilation of aromatic hydrocarbons, several strains were found to produce a large amount of cumic acid from p-cymene.

Five strains, S449B1, B2, B3, B4 and B6, were isolated from soil with the aromatic hydrocarbon substrates. They all assimilated both p-cymene and cumene. The strain S449B3 grew also on p-xylene, and S449B6 on p-xylene, toluene, and ethylbenzene.

They were all shown to be capable of producing an ultraviolet-absorbing substance from p-cymene. This substance was isolated in crystalline form and identified as cumic acid by infrared absorption spectrum and other observations.

The superior strain, S449B6, produced the acid as much as 1000 mg/1 in shaking culture at 30°C after 24 hours. The yields were increased up to approximately 1700 mg/1 after further investigations. Addition of calcium carbonate and considerable agitation were favorable conditions for the acid production.

The taxonomical studies of these strains were carried out, and they were all identified as closely resembling Pseudomonas desmolytica.     

Group-wise growth of Streptomyces in a medium containing streptomycin

Summary We have described the observation that Streptomyces griseus colonies grow group-wise on agar media containing streptomycin. We have found that this phenomenon is due to a substance (s) produced by germinating Str. griseus spores in media containing streptomycin, and this substance made the neighbouring spores more tolerant to increasing streptomycin concentrations. The substance is produced specifically by Str. griseus strains. The substance has probably a great molecular size, is thermolabile, not a nucleic acid and the applied enzymes did not inactivate it. Some investigated enzyme-poisons did not influence either its production or its effect on Str. griseus spores. We succeeded in carrying over the substance into liquid phase and separate it from the producing culture and this enables us to furterh purification and investigation of the substance.     

Isolation of Endophytic Actinomycetes From Roots and Leaves of Banana (Musa Acuminata) Plants and Their Activities Against Fusarium oxysporumf. sp. cubense

Two hundred and forty-two actinomycete strains were isolated from the interior of leaves and roots of healthy and wilting banana plants. Most of them were streptomycetes, Streptomyces griseorubiginosus-like strains were the most frequently isolated strains. Community analysis demonstrated increased actinomycete diversity in wilting leaves compared to that in healthy leaves, similar actinomycete communities were found in wilting and healthy roots. Screening of the isolates for antagonistic activity against Fusarium oxysporumf. sp. cubenserevealed that the proportion of antagonistic streptomycetes in healthy roots was higher than that in wilting roots (P < 0.01), but no difference was found between antagonistic strains isolated from healthy and wilting leaves. The potential biological control of Panama disease of banana by endophytic streptomycetes, especially Streptomyces griseorubiginosus-like strains was discussed.     

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.     

Isolation and Identification of α,α-Trehalose and Glycerol from an Arctic Psychrotolerant Streptomyces sp. SB9 and their Possible Role in the Strain's Survival

Abstract

Streptomyces sp. strain SB9 was isolated from perm frost soil samples in Spitsbergen, Arctic Ocean; it grows in a temperature range between 4°C and 28°C. During the survey of biologically active metabolites biosynthesized by this strain, significant amounts of α,α-trehalose (1) and glycerol (2) were detected. The compounds were isolated from the mycelium, were chromatographically separated, and the structures were elucidated on the basis of MS and NMR measurements. A possible role of trehalose in cold adaptation of the strain was examined. It was determined that the mycelium of the strain cultivated at 4°C accumulated 5-fold higher amounts of trehalose in comparison with the cells cultivated at 28°C. The mesofilic reference strains, Streptomyces spectabilis NRRL 2494 and Streptomyces lividans TK64, accumulated 100-fold less trahalose than the psychrotolerant Streptomyces sp. SB9. High amounts of trehalose in the cells could be a reason for adaptation of the strain to life at Arctic conditions.     

PCR Detection of Oxytetracycline Resistance Genes otr(A) and otr(B) in Tetracycline-Resistant Streptomycete Isolates from Diverse Habitats

A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (Tc^R) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of Tc^R streptomycetes originated from bulk and rhizosphere soil. Fewer Tc^R streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene–carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which Tc^R streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common.     

Evolutionary relationship of the Bacillus licheniformis macrolide-lincosamide-streptogramin B resistance elements

Summary Naturally occurring erythromycin (Em) resistance was found in 11 of the 18 Bacillus licheniformis isolates tested but was absent from a wide variety of other Bacillus strains. The Em resistance elements confer inducible macrolide-lincosamide-streptogramin B (MLS) resistance and are related to ermD an MLS resistance element previously cloned from the chromosome of B. licheniformis 749. The MLS sensitive B. licheniformis strains and the other sensitive Bacillus strains tested, lack sequences with detectable homology to ermD. The sensitive B. licheniformis strains do exhibit homology to sequences which flank ermD in B. licheniformis 749. The relative sizes of the homologous DNA fragments suggest that the sensitive strains are lacking a 3.6 kb segment which contains ermD. It is shown that ermD is homologous to chromosomal DNA from Streptomyces erythreus ATCC 11635, an Em producing organism. These observations suggest to us that MLS resistance may have arisen in the Streptomyces and spread to B. licheniformis another gram positive bacterium found in soil. It is further proposed that ermD is or was located on a transposon-like element and has spread and evolved further to yeild a variety of related Staphylococcal and Streptococcal MLS determinants.     

Use of mutagens in the improvement of production strains of microorganisms

Physical and chemical agents were employed in our laboratories to induce mutation in a variety of microorganisms used for production of antibioties or enzymes. Improved production strains ofPenicillium chrysogenum (penicillin-producer),Streptomyces griseus (streptomycin-producer),Streptomyces nodosus. (amphotericin B-producer),Streptomyces noursei (nystatin-producer),Streptomyces umbrinus (diumycin-producer),Streptomyces prasinus (prasinomycin-producer),Streptomyces roseochromogenes (steroid-16α-hydroxylase-producer), andArthrobacter simplex (steroid-1-dehydrogenase-producer) were developed by use of mutation selection techniques. The methods found to be most successful with each species are described. The genealogical relationships within species of a number of strains ofPenicillium chrysogenum, Streptomyces prasinus, andStreptomyces roseochromogenes are presented.     

Genetic divergence among rice strains

Summary For screening better types among 10 strains with the help of the D ² statistic, an experiment was conducted in randomized block design with three replications. 10 plants in each plot were randomly selected and observations were recorded on final plant height, heading duration, effective tillers, number of grains per panicle, yield per plant and 100 grain weight.The observations were analyzed and found to be significant at 1% level.In the study of distance relations the contribution of grain number per panicle to D ² values was found to be maximum. Again, this technique also helped in grouping the 10 strains into four clusters, viz. A, B, C and D, of which B possessed six strains out of 10; A possessed two and C and D one each.Group A comprised 2 selections from the same cross. Group D was composed of a selection from a cross involving Dular and Taichung Native-1 as parents. Group B possessed Dular, two selections from a cross involving Dular as one of the parents, Dharial and N.C.-1626. The constituents of A are expected to be transgressive segregates.Group A was maximally distantly related with D followed in order by C and B. The distance between B and C was small.Group D was found to be the best of all strains studied, followed by B, C and A.Dedicated to Prof. J. Straub on the occasion of his 60th birthday.     

Chemical Characterization of Lipid A from Some Marine Proteobacteria

Lipids A from type and wild strains of marine Proteobacteria belonging to Alteromonadaceae (Alteromonas (1 species), Idiomarina (1 species), and Pseudoalteromonas (8 species) genera) and Vibrionaceae (Shewanella (1 species) and Vibrio (1 species) genera) families and Marinomonas genus (1 species) were isolated by hydrolysis of their respective lipopolysaccharides with 1% acetic acid. Based on thin-layer chromatography data, the lipids A studied had low heterogeneity and generated family-specific patterns varying in numbers of bands and their chromatographic mobility. Total chemical analysis of the compounds showed that they contained glucosamine, phosphate, and fatty acids with decanoate (I. zobellii KMM 231^T lipid A) or dodecanoate (lipids A of the other bacteria) and 3-hydroxy alkanoates as the major fatty acid components. Unlike terrestrial bacterial lipids A, lipids A of marine Proteobacteria had basically monophosphoryl (except V. fluvialis AQ 0002B lipid A with its two phosphate groups) and pentaacyl (except S. alga 48055 and V. fluvialis AQ 0002B lipids A which were found to have six residues of fatty acids per molecule of glucosamine disaccharide) structural types, low toxicity, and may be useful as potential endotoxin antagonists.     

Urocanic Acid,Produced by Sporogenous Bacteria

On the screening of microorganisms which accumulate ultra violet light absorbing substances, some sporogenous bacteria were selected as powerful strains for production of a crystalline substance having a maximum absorption at 267.5 m_μ (pH 6.0 in water). These strains were microbiologically examined and named B. subtilis var. thermophilus. Submerged fermentation was carried out for 48 hrs at 37°C in glucose bouillon medium and the isolation of substance was performed by absorption to active carbon and elution with ammonia water.

On the basis of chemical studies of this crystal, it was identified as urocanic acid.