A New Regulatory Function of A-Factor: Stimulation of the Germination of Streptomycete Spores

Spore germination in streptomycetes was shown to be stimulated by exogenously added A-factor. Agar medium either containing or not containing A-factor was inoculated with spore suspensions of three strains differing in their ability to produce regulators of the A-factor group: Streptomyces griseus 773, which produces A-factor and two its lower homologs; S. coelicolor A3(2), which forms six Acl-factors (A-factor analogues); and S. avermitilis JCM5070, which fails to form regulators of this group. A count of the grown colonies showed that exogenous A-factor stimulated spore germination in strains that were themselves able to synthesize regulators of the A-factor group. In S. griseus 773, the number of germinated spores increased by 67% on average after the addition of A-factor to the medium in an amount of 10 g/ml. In strain S. coelicolor A3 (2), the number of germinated spores increased by 75% after the addition of 1 g/ml of A-factor. During germination of the S. avermitilis JCM5070 spores, no changes in the CFU number was observed after the addition of A-factor.     

Screening of compounds stimulating spore formation and mycelial growth of pock-forming plasmid-carrying strains in Streptomyces azureus

Summary Sporulation-stimulating compounds were screened by using mutant PK100a of Streptomyces azureus ATCC 14921, in which spore formation was markedly inhibited by the pock-forming plasmid pSA1.1. On agar media, cysteine, bacitracin, glutathione and -NAD induced the formation of coloured spores or spore mass of strain PK100a. These compounds also stimulated the spore formation of the wild-type and its plasmid-cured (good spore-forming) strains and the growth of aerial mycelia of these three strains. This screening method appears to be an effective method for the screening of substances that stimulate spore formation and mycelial growth of streptomycetes or that overcome the inhibitory effect of pock-forming plasmids. Correspondence to: S. Ogata     

Biosynthesis of Streptomycin

Investigation was carried out on demonstration of two substances constructing a precursor system located at a late stage of streptomycin biosynthesis by Streptomyces griseus. One of them is thought to be a natural precursor of Streptomycin(L) and the other is suggested as an enzymatic substance(H) transforming L to streptomycin. Both substances had no antibiotic potency and H was inactivated at low pH. L was obtained from a cell-free supernatant (active supernatant) prepared from suspension of young mycelium of Streptomyces griseus in glucose solution. H was obtained not only from active supernatant but also from cell-free extract of the organism.

Two ways of isolation were established for L. Active supernatant was adsorbed on a CM-cellulose column equilibrated with 0.05 m Tris-maleate buffer (pH 8.0). Elution of this column with the same buffer as was used for equilibration gave L-containing fraction separated from streptomycin which was eluted with the buffer including 1% of sodium chloride. L was adsorbed also on active carbon in aqueous solution at neutral pH and liberated from it at acidic pH with 95% methyl alcohol. The former method was useful to separate L from streptomycin, and the latter one was so to concentrate L.

H was isolated by using a column chromatography on DEAE-cellulose. After adsorbing active supernatant or cell-free extract of organism on a column equilibrated previously with the same buffer as above, H was eluted with the buffer including 1% of sodium chloride. Cell-free extract of S. griseus was a better source of H supply than the active supernatant.     

Bacteriolytic Activity of Enzymes Derived from Streptomyces Species

Streptomyces species H–402 and 1829 possessing high lytic activities against cariogenic streptococci which induce dental plaque and caries, were isolated by the screening from soils and sewers. They were identified as Streptomyces griseus and Streptomyces globisporus respectively. The former strain produced lytic enzyme accompanying spore formation during the surface culture, while the latter strain revealed a high activity in the submerged culture. These enzymes had wide substrate specificity against all groups of cariogenic streptococci. The lytic enzymes may be expected as an useful medicament for the prevention of dental caries.     

Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces.

Summary A 7.2 kbBglII restriction fragment, which increases the production of several extracellular enzymes, including alkaline phosphatase, amylase, protease, lipase and -galactosidase, was cloned inStreptomyces lividans from the DNA ofS. griseus ATCC 10137. This gene (namedsaf) showed a positive gene dosage effect on production of extracellular enzymes. When thesaf gene was introduced into cells in high copy numbers it delayed the formation of pigments and spores inS. lividans and also retarded actinorhodin production inStreptomyces coelicolor. Thesaf gene hybridized with specific bands in the DNA of severalStreptomyces strains tested. A 1 kb fragment containing thesaf gene was sequenced and contains an open reading frame (ORF) of 306 nucleotides which encodes a polypeptide of M_r 10 500. This ORF is contained within a fragment of 432 by which retained activity inStreptomyces. A fragment with promoter activity is present upstream of thesaf reading frame. The predicted Saf polypeptide has a strong positive charge, and does not show a typical amino acid composition for a membrane protein, and contains a DNA-binding domain similar to those found in several regulatory proteins.     

Transient gene expression in electroporated Picea glauca protoplasts

The reporter gene for chloramphenicol acetyltransferase (CAT) was introduced into white spruce (Picea glauca (Moench) Voss.) protoplasts by electroporation. CAT transient gene expression was increased by increasing the concentration of pCaMVCN plasmid and was affected by the level of the applied voltage. Highest CAT activities were obtained after electroporation with a pulse of 350V.cm^–1 having an exponential decay constant of approximately 105ms. Linearized plasmid constructs gave much higher levels of CAT activity than circular plasmid. Attempts to use the Escherichia coli -glucuronidase gene (-GUS) as a marker gene revealed very high levels of -GUS-like activity in electroporated protoplasts. This activity was mainly due to a small molecule and may mask successful transformation since -GUS-like activity increased when plasmid DNA was present during electroporation.Abbreviations CAT chloramphenicol acetyltransferase - -GUS -glucuronidase - MUG 4-methyl umbelliferyl glucuronide - F microfarads NRCC No. 29150     

Truncated presequences of mitochondrial F1-ATPase β subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco

The mitochondrial F₁-ATPase subunit (ATPase-) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH₂-terminal extension. By sequencing the mature N. tabacum ATPase-, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3 deletions in the sequence coding for the 90 NH₂-terminal residues of ATPase-. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and -glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase- remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.     

A respiratory nitrate reductase active exclusively in resting spores of the obligate aerobe Streptomyces coelicolor A3(2)

The Gram‐positive aerobe Streptomyces coelicolor undergoes a complex life cycle including growth as vegetative hyphae and the production of aerial hyphae and spores. Little is known about how spores retain viability in the presence of oxygen; however, nothing is known about this process during anaerobiosis. Here, we demonstrate that one of the three respiratory nitrate reductases, Nar‐1, synthesized by S. coelicolor is functional exclusively in spores. A tight coupling between nitrite production and the activity of the cytoplasmically oriented Nar‐1 enzyme was demonstrated. No exogenous electron donor was required to drive nitrate reduction, which indicates that spore storage compounds are used as electron donors. Oxygen reversibly inhibited nitrate reduction by spores but not by spore extracts, suggesting that nitrate transport might be the target of oxygen inhibition. Nar‐1 activity required no de novo protein synthesis indicating that Nar‐1 is synthesized during sporulation and remains in a latently active state throughout the lifetime of the spore. Remarkably, the rates of oxygen and of nitrate reduction by wetted spores were comparable. Together, these findings suggest that S. coelicolor spores have the potential to maintain a membrane potential using nitrate as an alternative electron acceptor.     

Activation and analysis of crypticcrt genes for carotenoid biosynthesis fromStreptomyces griseus

Genes encoding enzymes with sequence similarity to carotenoid biosynthetic enzymes of other organisms were cloned fromStreptomyces griseus JA3933 and transformed into the colourless (non-daunorubicin producing) mutantStreptomyces griseus IMET JA3933/956/2. Cells harbouring these genes showed an orange-red pigmentation, caused by the strongly hydrophobic, membrane-bound lycopene. The cloned fragment (9 kb) contained seven genes, four transcribed in one direction (crtEIBV) and three (crtYTU) transcribed convergently to them. Three of these genes encode polypeptides that resemble geranylgeranyl-pyrophosphate (GGPP) synthases (CrtE), phytoene synthases (PS) (CrtB) and phytoene dehydrogenases (PDH) (CrtI), respectively, of various bacteria. These enzymes are sufficient for the formation of lycopene.crtE alone was sufficient to induce zeaxanthin formation in anEscherichia coli clone containing thecrt gene cluster fromErwinia herbicola deleted forcrtE. The combination ofcrtE andcrtB led to formation of phytoene inS. griseus. The putativecrtEp promoter region was cloned and mapped by primer extension analysis. In a gel retardation experiment, this fragment was specifically shifted by an unknown protein. CrtY shows similarity to lycopene cyclases that convert lycopene into-carotene, CrtT resembles various methyltransferases and CrtU a dehydrogenase. We conclude that these genes are functionally intact, but not expressed (cryptic) in the wild-typeS. griseus strain.     

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.     

Demonstration of the presence of an inducibleβ-lactamase inAzospirillum lipoferum

Azospirillum lipoferum is a soil microorganism that has been shown to be resistant to penicillins. It has been suggested that resistance is due to the presence of a -lactamase activity, but no conclusive evidence has been reported. The incubation of benzylpenicillin, or nitrocephin with either wholeAzospirillum cells or cell-free extracts was accompanied, by hydrolysis of the -lactam ring of the antibiotics. Such hydrolytic activity exhibited Michaelis and Menten-like kinetics. The enzyme was produced at a low, basal level that was increased approximately 50 times by the addition of benzylpenicillin, an increase that was completely blocked by chloramphenicol or rifampicin.     

Factors affecting transient gene expression in electroporated Glycine max protoplasts

Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 F and a protoplast concentration of 1–3 × 10⁶/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - PEG polyethylene glycol     

Transient expression in electroporated pea protoplasts: Elicitor responsiveness of a phenylalanine ammonia-lyase promoter

Summary High yields of viable pea protoplasts were produced from suspension cultured cells and the conditions for the optimum transient expression of the chloramphenicol acetyltransferase (CAT) gene fused to the CaMV 35S promoter after electroporation were investigated. Conditions for elicitor induction of a member of the phenylalanine ammonia-lyase (PAL) gene family in pea was also investigated using a chimeric gene carrying 480 bp of the putative promoter region of gPAL1 connected to bacterial cat gene and nos terminator. CAT activity was considerably induced by the treatment with fungal elicitor (>100 g/ml glucose equivalent) isolated from Mycosphaerella pinodes, a pea pathogen.Abbreviations CAT chloramphenicol acetyltransferase - PAL phenylalanine ammonia-lyase - CM acetylated chloramphenicol - GSH reduced glutathione - NOS nopaline synthase - ES electroporation solution - CaMV cauliflower mosaic virus - GUS -glucuronidase - CHS chalcone synthase - 2,4-D 2, 4-dichlorophenoxyacetic acid Present address: Research Institute, Takasago Perfumery Inc, 5-31-36, Kamata, Minato, Tokyo, 144, Japan Toso Inc, 4560 Oaza-Tomita, Shin-nanyo, Yamaguchi, 746, Japan Central Laboratory of Green Complex, Kasetsert University, Kamphaensaen, Nakohn Pathom, Thailand     

Zur Kenntnis thermophiler Actinomyceten

Zusammenfassung Für Thermomonospora curvata und Streptomyces rectus=Actinomyces thermophilus sensu Gilbert werden Neotypen vorgeschlagen. Für Streptomyces thermoviolaceus ssp. thermoviolaceus und ssp. apingens, S. thermovulgaris, Pseudonocardia thermophila und Microbispora bispora werden Typusstämme ausgewählt. Thermomonospora curvata wird als Typusart für die Gattung Thermomonospora gewählt. Die Gattung Thermopolyspora wird Synonym von Microbispora. Von den Thermopolyspora-Arten kommt nur Th. bispora zur Gattung Microbispora. Für Th. polyspora, Th. flexuosa und Th. rectivirgula ist später eine neue Gattung aufzustellen.Folgende Sporenfeinstruktur wurde für die thermophilen Arten festgestellt: Bei Streptomyces thermovulgaris, Pseudonocardia thermophila und Microbispora bispora sind die Spore glatt, sie sind stachelig bei Streptomyces rectus und Thermomonospora curvata und warzig bei Streptomyces thermoviolaceus. Die Warzen sind in der Mitte vertieft.Ultradünnschnitte wurden von den Sporen von Thermomonospora curvata hergestellt. Die Stacheln werden durch die Falten einer Hülle gebildet, welche locker die dickwandige, glatte Spore umgibt. Die Ähnlichkeit der Bilder mit Microellobosporia ist vielleicht ein Hinweis auf eine mögliche Verwandtschaft der beiden Gattungen.Bei Pseudonocardia thermophila wurden das Mycelwachstum und die Sporenbildung im einzelnen verfolgt. Sowohl die Substrat- als auch die Lufthyphen sin septiert. Viererlei Sporen werden gebildet: Zerfallssporen im Substratmycel, Zerfallssporen im Luftmycel, Segmentationssporen in den Lufthyphen=Entstehung nach dem Pseudonocardia-Typ und Sporenbildung in den Lufthyphen nach dem Streptomyces-Typ.Fünf Formen der Luftsporenbildung werden diskutiert: der Streptomyces-Typ, der Microbispora-Typ, der Thermomonospora-Typ, der Pseudonocardia-Typ und die Sporenbildung, wie sie Krassilnikov u. Agre für Thermopolyspora-Arten beschrieben.

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Studies on thermophilic actinomycetes

Summary Typecultures are designated for the species Streptomyces thermoviolaceus ssp. thernoviolaceus and ssp. apingens, S. thermovulgaris, Pseudonocardia thermophila and Microbispora bispora; neotypes are proposed for Thermonospora curvata and Streptomyces rectus=Actinomyces thermophilus sensu Gilbert. Thermonospora curvata is chosen as type species of the genus Thermonospora. The genus Thermopolyspora becomes a synonym of Microbispora. A new genus will have to be erected for Thermopolyspora polyspora, Th. flexuosa and Th. rectivirgula, which do not fit into the genus Microbispora.The fine structure of the spores of some thermophilic species is described. Streptomyces thermovulgaris, Pseudonocardia thermophila and Microbispora bispora have smooth spore walls; spines are developed in Streptomyces rectus and Thermomonospora curvata, and warts in Streptomyces thermoviolaceus. The warts have been found to possess a median depression.Ultrathin sections were cut of the spores and hyphae of Thermomonospora curvata. The spine bearing layer forms a loose sheath around the smooth, thick wall of the spore as is reported in Microellobosporia. This may indicate relationship between these two genera.In Pseudonocardia thermophila the development of the substrate and aerial mycelia and the spore formation has been studied in detail. Both the substrate and aerial hyphae are septate. Four kinds of spores were observed: fragmentation spores in the substrate hyphae, fragmentation spores in the aerial hyphae, segmentation spores in the aerial hyphae produced according to the Pseudonocardia-type, and spores formed in the aerial hyphae according to the Streptomyces-type.Among the non-sporangiate actinomycetes five types of aerial spore formation are discussed, the Streptomyces-type, Microbispora-type, Thermomonospora-type, Pseudonocardia-type, and the formation of spores described by Krassilnikov and Agre for two species of Thermopolyspora.

     

Morphological characterization and germination of aerial and submerged spores of the entomopathogenic fungus Verticillium lecanii

Under solid-state and liquid-state cultivations, the entomopathogenic fungus Verticillium lecanii F091 produced different types of spores. The aerial spores (AS) on cooked rice formed clusters on the tips of conidiophores, while the submerged spores (SS) were dispersed in the medium. The aerial spore appeared relatively uniform in size, which was 6.1 ± 0.9 m long, and 2.2 ± 0.3 m wide. The submerged spore varied in shape and size, with a mean length of 5.0 ± 1.0 m and width of 1.9 ± 0.5 m. Under scanning electron microscopy, the AS had a tendency to have rough, brittle surface characteristics; however, the SS appeared smooth on the surface. These spores were compared in two different germination media. On SMAY (Sabouraud maltose, agar, yeast extract, and neopeptone) coated coverslips, the AS did not show germ tubes until 8 h of incubation; while the SS showed many germ tubes. However, over 90% spore germination ratio was reached for both types of spores at 18-h of incubation. In the liquid medium, the SS germinated rapidly and many spores even produced spores on the spores; while the AS germinated, grew, and branched in the submerged culture gradually, and some sporulated on the tips of the short branches, or on the mycelia until 18 h of incubation. Evidently, the germination, growth patterns of aerial or submerged spores differed greatly under the different culture conditions. The virulence of the pathogen in relation to the type of spore of V. lecanii is discussed.     

Relationship between macrotetrolide production and specific activity of some hydrolases in a high and a low producing strain of Streptomyces griseus

The activities of five hydrolytic enzymes in the culture filtrate and in cell-free extracts from strains of Streptomyces griseus, differing in macrotetrolide production, have been determined over a fermentation period of 200 h. The specific activities of phosphatase, phosphodiesterase, and adenosine triphosphatase in the medium, and phosphatase and phosphodiesterase in the cell-free extract were lower in the low than in the high producing strain. No significant difference was found between the strains, for adenosine triphosphatase and protease activity in the cell-free extract or protease activity in the medium. The specific activity of esterase was higher in the low than in the high producing strain.     

Parameters affecting the activity of antisense RNA sequences in tobacco protoplasts

Summary Plasmids containing various fragments of the -glucuronidase (GUS) gene were placed in antisense orientation downstream of the cauliflower mosaic virus 35S promoter and cotransfected with a 35S-gus construct into tobacco mesophyll protoplasts. None of the partial-length sequences were as effective as the full-length sequence in reducing GUS activity. The presence of a polyadenylation sequence downstream of the antisense sequence had an enhancing effect. The activity of the antisense sequence was largely affected by the incubation temperature of the transfected protoplasts. The chloramphenicol acetyltransferase (CAT) gene was fused to the gus coding sequence. When this construct was cotransfected with an antisense sequence directed against CAT, GUS activity was reduced. The implications of these results for the design and uses of antisense sequences are discussed.     

Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles

Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F₁-ATPase -subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and -glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial -presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immuno-detection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import.     

The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

Microbodies (peroxisomes) in the toad,Bufo marinus

Summary Microbodies (peroxisomes), a group of cytoplasmic organelles enriched in catalase, are demonstrated in the toad, Bufo marinus, by light and electron microscopy by means of a cytochemical staining procedure that demonstrates the peroxidatic activity of catalase with diaminobenzidine (DAB). Amphibian microbodies are similar to those of other classes in their fine structure and localization in hepatocytes and kidney, where they are prominent in the proximal tubular cells. Nucleoids are present only in renal microbodies. In the proximal renal tubule an unusual group of large brown granules are identified as lysosomes by their acid phosphatase, -glucosaminidase and -glucuronidase activities.This work was supported by U.S. Public Health Service Grants Nos. NS-06856 and HD 00674. We wish to thank Dr. Richard M. Hays who generously supplied us with toads; Dr. Alex B. Novikoff for making available facilities for ultramicrotomy, Miss Betty De Prest for technical assistance; Miss Marianne Van Hooren for preparation of the photomicrographs.