Improved yields of daunomycinone glycosides in developmental mutants ofStreptomyces coeruleorubidus

When improvingStreptomyces coeruleorubidus JA 10092, a producer of antibiotics of the daunomycinone complex, the most active variants were found among isolates of morphological typesbld-1 (with a suppressed production of the aerial mycelium on organic media containing glucose) andwhi (with an asporogenic aerial mycelium on glucose media and with the bald phenotype on media containing starch). Submerged cultures of thewhi mutants produced increased quantities of daunomycinone glycosides in the antibiotic complex, the amount of free anthracyclinones being simultaneously decreased. Thewhi strains differed from the wild type also in higher demands for aeration, concentration of glucose and in an increased production capacity in starch media. The overall antibiotic activity increased more than 40 times after a six-step selection (application of UV light, γ-radiation, nitrous acid and natural spreads) combined with an altered fermentation technology.     

Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor

In wild-type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3′,5′ monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse-labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ-tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross-feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 μM) found in the medium of MT1110 cultures, suggested that it may serve as a diffusible signalling molecule to co-ordinate antibiotic biosynthesis.     

A surface active protein involved in aerial hyphae formation in the filamentous fungus Schizophillum commune restores the capacity of a bald mutant of the filamentous bacterium Streptomyces coelicolor to erect aerial structures

The filamentous bacterium Streptomyces coelicolor undergoes a complex process of morphological differentiation involving the formation of a dense lawn of aerial hyphae that grow away from the colony surface into the air to form an aerial mycelium. Bald mutants of S. coelicolor, which are blocked in aerial mycelium formation, regain the capacity to erect aerial structures when exposed to a small hydrophobic protein called SapB, whose synthesis is temporally and spatially correlated with morphological differentiation. We now report that SapB is a surfactant that is capable of reducing the surface tension of water from 72 mJ m^?2 to 30 mJ m^?2 at a concentration of 50 μg ml^?1. We also report that SapB, like the surface-active peptide streptofactin produced by the species S. tendae, was capable of restoring the capacity of bald mutants of S. tendae to erect aerial structures. Strikingly, a member (SC3) of the hydrophobin family of fungal proteins involved in the erection of aerial hyphae in the filamentous fungus Schizophyllum commune was also capable of restoring the capacity of S. coelicolor and S. tendae bald mutants to erect aerial structures. SC3 is unrelated in structure to SapB and streptofactin but, like the streptomycetes proteins, the fungal protein is a surface active agent. Scanning electron microscopy revealed that aerial structures produced in response to both the bacterial or the fungal proteins were undifferentiated vegetative hyphae that had grown away from the colony surface but had not commenced the process of spore formation. We conclude that the production of SapB and streptofactin at the start of morphological differentiation contributes to the erection of aerial hyphae by decreasing the surface tension at the colony surface but that subsequent morphogenesis requires additional developmentally regulated events under the control of bald genes.     

Influence of the culture medium composition on the excreted/secreted proteases from <Emphasis Type="Italic">Streptomyces violaceoruber</Emphasis>

Streptomyces violaceoruber produces two different classes of mycelium, the substrate and the aerial mycelium. Since proteases have been associated with morphological turnover processes in other Streptomyces species, the presence of excretory/secretory proteolytic activities was investigated here in S. violaceoruber culture supernatants. Various polypeptide bands, with apparent molecular masses ranging from 40 to 180 kDa, were detected in soy trypticase broth (STB) culture media supernatants following 72 h of growth, using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Zymograms showed the presence of five proteolytic enzymes (Spvio1–5), which migrated as bands of 167.7, 130.7, 110.7, 48.3 and 40.9 kDa, respectively. The characterization of these proteases by specific inhibitors showed that Spvio1–4 belong to the serine protease group and Spvio5 corresponds to a cysteine protease. Additionally, Spvio2 and 5 were inhibited by a mixture of EDTA and EGTA, indicating that both require divalent cations. The protease pattern obtained in STB enriched with glucose was identical to that obtained in STB. However, Spvio3 and 4 were absent when nitrogen was added to the culture medium. Cell death was fluorescently detected following 72 h of S. violaceoruber growth in STB and in STB that was enriched with glucose. On the contrary, no cell death was detected in nitrogen-enriched STB media. Additionally, the formation of the aerial mycelium was impaired in solid cultures of STB media enriched with nitrogen. These results demonstrate that the composition of the media influences the morphological turnover of the colony and the pattern of excreted/secreted proteases from S. violaceoruber, and suggest that Spvio3 and 4 are involved in the aerial mycelium formation.     

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

A preliminary study of the applicability of sweet potatoes for mycological fat production

Summary For mycological fat production,Aspergillus nidulans andPenicillium lilacinum were grown on media made up of crushed and boiled sweet potatoes. Mycelial mats containing a fair amount of fat were obtained, namely, 15 % in case ofAspergillus and 24 % in case ofPenicillium. Media containing an external supply of glucose gave rise to increase in percentage fat in mycelium, but the amount of mycelium and consequently the total fat content decreased.Addition of sodium nitrate gave heavier mycelial mats but the total fat content could not be raised.Addition of potassium phosphate or magnesium sulphate or both to the sweet potato medium gave rise to heavier mycelial mats but with lower fat content.     

Fusarium nisikadoi, a new species from Japan

A new species ofFusarium, F. nisikadoi, isolated fromPhyllostachys nigra var.henonis (bamboo) andTriticum aestivum (wheat) in Japan, is described, illustrated and discussed. This species is differentiated from other known species of the genus by the following characteristics: whitish colony color, long zigzag-like chains of 0–3(-5)-septate clavate conidia, intermixed with pyriform conidia, produced mostly from monophialides and rarely from polyphialides in the aerial mycelium, very long and slender sporodochial conidia, and no chlamydospores. The long chains of septate conidia are known only in this species of the genusFusarium. The conidiophores on the aerial mycelium sometimes proliferate sympodially. The species is tentatively placed in the form-sectionLiseola.     

Evidence of heterokaryosis in streptomyces coelicolor A3(2)

Summary Hyphae from mixed cultures of complementary auxotrophs of Streptomyces coelicolor A3(2) did not grow on minimal media (MM) when fertility plasmids (SCP1 and SCP2) were missing in both strains. The addition of one part per cent of complete medium (CM) to MM allowed growth of vigorous colonies among the tiny colonies of the parental types. The former, amounting to 1%–10% of the total population, turned out to be heterokaryons. They could be propagated on the same medium by plating of hyphal fragments. When five parts per cent of CM were added to MM, beside the heterokaryotic colonies vigorous spindles of aerial mycelium were formed whenever complementary colonies overlapped. When the SCP1 and SCP2 plasmids were present in one or both parents a clear constraint on the outburst of heterokaryotic aerial mycelium was observed.     

Hyphal and mycelial interactions betweenAgaricus bisporus andScytalidium thermophilum on agar media

The interaction betweenAgaricus bisporus andScytalidium thermophilum on agar media was studied by differential interference contrast and phase contrast microscopy.A. bisporus combatively replacesS. thermophilum in culture on agar media. The antagonistic effect ofA. bisporus is transmissible through a cellophane membrane and causes irreversible disintegration ofS. thermophilum protoplasm, resulting in a total loss of viability after prolonged interaction between the two fungi. On compost extract agar, but not on other media, the growth rate ofA. bisporus increased from 2.7 to 5.3 mm·d^–1 following contact withS. thermophilum mycelium.     

Age-dependent differential responses ofSaprolegnia hyphal tips to a helical growth-inducing factor in the agar substitute,gellan

Saprolegnia ferax produces more-or-less straight, subapically branched, hyphae when growing in liquid or agar-solidified media, with abundant aerial mycelium on the latter. In Contrast, the same medium solidified with gellan gum induced helical growth with reduced branching and almost no aerial mycelium. Helical growth induction was gellan concentration-dependent, peaking at 0.4–0.6% (w/v), when about 60% of tips were helical. Gellan-induced helices showed concentration-dependent inhibition by agarose and polyethylene glycol. Colonies on gellan-agarose, where helices were inhibited, reverted to having aerial mycelium, whereas those on gellan-polyethylene glycol did not. Branches on helical hyphae were initially linear, but converted to helical growth after about 2 h of extension. This transition was often marked by a branch, thus branch and helix competency appeared to be related. Germinating cysts took twice as long as hyphal inocula before producing helical hyphae, reinforcing the suggestion that helix competence was age-related.Achlya, but notPhytophthora, also showed gellan-induced helical growth and aerial mycelium suppression. These results showed (a) that morphogenic regulators of hyphal growth responded to gelling agents, probably high-molecular-weight polysaccharides, (b) that all growing hyphal tips were not equivalent, and (c) that hyphal tips underwent age-related changes in their response to the environment. The gellan-related differences in aerial mycelium mimic hydrophobin-based mycelium behavior and may thus indicate environmental regulation of hydrophobin production.     

Production of gluconic acid by immobilized in pumice stones mycelium of Aspergillus niger using unconventional oxygenation of culture

Gluconic acid was produced in repeated batch processes with Aspergillus niger AM-11, immobilized in pumice stone particles using an unconventional oxygenation of culture media based on the addition of H₂O₂, decomposed by catalase to O₂ and water. The highest gluconic acid productivity of 8.2 g l^–1 h^–1 was reached with 30 g immobilized mycelium per 150 ml, 10% (w/v) glucose, at 24 °C and pH 6.5, with O₂ at 100% saturation. The immobilized mycelium was successfully reused up to 8 times in 1-h batches with only a slight loss (11%) of gluconic acid productivity.     

Utilization of carbohydrates by Helminthosporium rostratum drechs. and Deightoniella torulosa (SYD). Ell. (SYN. Helminthosporium torulosum SYD.)

Summary Utilization of four carbohydrates, viz. D-glucose, D-fructose, sucrose and starch, byHelminthosporium rostratum andDeightoniella torulosa isolated from the leaves ofJasminum arborescens Vern. Newari and banana (Musa paradisiaca L.) fruits respectively, was exhaustively investigated by chromatographic technique. Chromatographic analysis of the culture medium revealed that both the organisms assimilated glucose earlier than fructose. The hydrolytic products of sucrose and starch (only glucose was detected) could be traced in the medium. In all cases dry weights of both the organisms continued to increase upto 15 days except inH. rostratum growing on starch where it decreased after 10th day. The pH changes of the media showed a drift towards neutrality.This research has been financed in part by a grant made by the United States Department of Agriculture, Agricultural Research Service, Under P.L. 480.     

Identification of bldA mutants of Streptomyces griseus

The bldA gene (encoding tRNA_UUA^Leu) from Streptomyces griseus (Sg) was cloned by hybridization with bldA from Streptomyces coelicolor (Sc). Introduction of Sg bldA into Sc bldA mutants restored sporulation and actinorhodin production. Sporulation of a subset of Sg bald mutants, which produce no aerial mycelium or spores, was restored in the presence of bldA from Sc or Sg. The nucleotide sequences of the bldA alleles from two such bald mutants revealed point mutations in the anticodon stem and the TΨC stem.     

Ability of ectomycorrhizal fungi to utilize starch and related substrates

Basidiomycetous fungi of 55 strains of 33 species in 15 genera which are thought to be ectomycorrhizal were grown on starch and related substrates as a sole carbon source, and their ability to utilize these substrates was determined. Mycellial weights of the fungi grown on agar media containing starch and amylose varied between the strains from 1.1 to 94.9 mg/flask and from 0.4 to 93.3mg/flask, respectively. Mycelial growth rates ranged from 0 to 1.17 mm/d on barley grain medium and from 0 to 2.03 mm/d on rice grain medium; the highest rate corresponded to about half of the average of reference wood-rotting fungi. Most of the mycorrhizal fungi that grew well on amylose gave higher growth rates on barley. Several strains inLyophyllum, Hebeloma, Sarcodon, andTricholoma grew well on both glucose and starch media.     

Induced tolerance of Sclerotium cepivorum to antibiotics in the presence of onion exudates

The onion white rot pathogen Sclerotium cepivorum was cultured on agar media containing 2% malt extract and one of the antifungal antibiotics, endomycin, griseofulvin, venturicidin and cycloheximide at concentrations that reduced but did not prevent growth of mycelium. When onion seeds or agar discs impregnated with diffusates from onion bulbs were placed on the antibiotic media, radial growth of the fungus was greatly increased, and there was a profuse development of aerial mycelium. Gaseous diffusates from onion tissue and from impregnated agar discs were also effective. On the antibiotic media, tomato, cabbage and radish seeds did not stimulate the growth of S. cepivorum and the onion exudates did not stimulate the growth of four other fungi. This and other evidence is considered to show that the stimulation of growth of S. cepivorum was not caused by any direct effect on the antibiotics but by a tolerance of the fungus to them, which was specifically induced by an exudate from its host plant. The phenomenon may be related to the reported reversal by onion extracts of the inhibitory effects of soil mycostasis on germination of sclerotia of the fungus.     

Hypothalamic GABA receptor functional regulation and liver cell proliferation

The influence of carbohydrate utilisation on the growth of three strains of Vittad. mycelium (1BO, 17BO and 10RA) in culture was assessed using culture media containing glucose (control), mannose or mannitol. Mannose was the best substrate for growth of the strains and this was particularly evident for strain 17BO. Mannitol instead was metabolized only by 10RA and 1BO. In order to explain the different growth trends, analyses of enzyme levels, kinetic parameters, protein patterns and the morphology of the three strains were carried out. Our results show that these strains of mycelium were affected by the substrates used in the media. The aim of the present work was to optimise the in vitro production of T. borchii mycelium for use in experiments which require the fungus in precise and reproducible conditions, such as mycorrhizal synthesis or protein and nucleic acid extractions.     

Study of the chemical makeup of the mycelium from an active strain of Act. rimosus and from an inactive mutant in relaiton to oxytetracycline biosynthesis]

Chemical composition of the mycelium of the active and inactive mutants of Act. rimosus grown under conditions favourable for oxytetracycline biosynthesis on the starch or maltose medium and under favourable conditions on the glucose medium was studied. It was shown that according to its chemical composition the above strains did not practically differ. When grown on the starch medium the mycelium of both strains contained great amounts of carbohydrates and comparatively small amounts of nucleic acids and nitrogen. Replacement of starch in the medium by glucose or maltose induced significant changes in the mycelium composition: the synthesis of intracellular polysaccharides was markedly suppressed and the synthesis of nucleic acids and nitrogen containing compounds increased. RNA was the main nucleic acid in both strains on starch and glucose media. The content of DNA was low and did not practically change. The mycelium of both strains contained small amounts of lipids which did not significantly change during the process of cultivation and did not correlate with the antibiotic activity.     

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.