Steroid transformations by species of Cephalosporium and other fungi

A total of 58 cultures, tentatively identified as species of the genus Cephalosporium, were screened in flask fermentations for their ability to effect conversions of progesterone (Delta(4)-pregnene-3,20-dione) and Reichstein's Substance S (Delta(4)-pregnene-17alpha,21-diol-3,20-dione). A large number of transformations were observed by means of a series of five paper chromatography systems rated for analysis of steroid compounds ranging in polarity from progesterone to polyhydroxylated steroids. Five different transformation products were selected for isolation and identification. For purposes of recovery, conversions were conducted under submerged conditions in either 4- or 200-liter fermentors in which the broth was agitated and aerated. The steroid substrate was dissolved in acetone and added aseptically to the growing culture in a final concentration of 0.025%. After the conversions were effected, the whole broth was extracted with chloroform, and the transformation products were recovered, either by direct crystallization from solvents or through the use of silica gel columns. It was determined that C. ciferrii 21C converted progesterone to Delta(4)-androstene-3,17-dione. Kendall's Compound F (Delta(4)-pregnene-11beta,17alpha,21-triol-3,20-dione) was converted to its 20beta-ol analogue by Geotrichum sp. 51C (during these studies, a number of cultures were taxonomically reclassified). Cephalosporium sp. 27C formed the Delta(1)-analogue of Reichstein's Substance S, and Cephalosporium sclerotigenum 31C and Verticillium aphidum both converted Substance S to the 6beta-hydroxy derivative. Paecilomyces persicinus 22C converted Substance S to a product believed to be a dihydroxylated derivative.     

Cephalosporium maydis is a distinct species in the Gaeumannomyces-Harpophora species complex

Cephalosporium maydis is an important plant pathogen whose phylogenetic position relative to other fungi has not been established clearly. We compared strains of C. maydis, strains from several other plant-pathogenic Cephalosporium spp. and several possible relatives within the Gaeumannomyces-Harpophora species complex, to which C. maydis has been suggested to belong based on previous preliminary DNA sequence analyses. DNA sequences of the nuclear genes encoding the rDNA ITS region, β-tubulin, histone H3, and MAT-2 support the hypothesis that C. maydis is a distinct taxon within the Gaeumannomyces-Harpophora species complex. Based on amplified fragment length polymorphism (AFLP) profiles, C. maydis also is distinct from the other tested species of Cephalosporium, Phialophora sensu lato and members of Gaeumannomyces-Harpophora species complex, which supports its classification as Harpophora maydis. Oligonucleotide primers for H. maydis were developed that can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen. These diagnostic PCR primers will aid the detection of H. maydis in diseased maize because this fungus can be difficult to detect and isolate, and the movement of authentic cultures may be limited by quarantine restrictions.     

Biosynthesis of penicillin N and cephalosporin C: Antibiotic production and other features of the metabolism of a Cephalosporium sp

1. The production of penicillin N and cephalosporin C by two mutants of a Cephalosporium sp. has been studied with cultures grown in a chemically defined medium and with suspensions of washed mycelium in water or a buffered salt solution. 2. Antibiotic synthesis began at an early stage of growth and its rate per unit weight of mycelium appeared to pass its maximum as morphological changes were occurring in young hyphae. This rate subsequently declined, but rapid production could continue after net growth had ceased. 3. In a series of shake-flask fermentations in the growth medium, increases in the yield of penicillin N above the mean were correlated with much smaller increases in the yield of cephalosporin C and vice versa. 4. In suspensions of washed mycelium, moderate decreases in the efficiency of aeration increased the yield of penicillin N and decreased that of cephalosporin C. A similar result normally followed the addition of methionine to the suspension fluid, and in both cases there was usually an increase in the yield of the two antibiotics combined. 5. The apparent intracellular concentrations of the antibiotics were much lower than those attained extracellularly and also much lower than those of most of the amino acids in the intracellular pool. No detectable amount of [(14)C]penicillin N added to the extracellular fluid was found to enter the mycelium. 6. Very small amounts of peptide material whose behaviour was similar to that of the sulphonic acid of delta-(alpha-amino-adipoyl)cysteinylvaline on paper electrophoresis at pH1.8 were found in extracts of the mycelium that had been oxidized with performic acid. 6-Aminopenicillanic acid and 7-aminocephalosporanic acid were not detected. 7. Ultrasonic treatment of the mycelium resulted in rapid fragmentation of mycelial chains, rupture of many individual cells, and the liberation of amino acids and other substances into the medium. 8. Ultrasonically treated preparations synthesized penicillin N and cephalosporin C rapidly after a lag of 12hr. Antibiotic synthesis was accompanied by the growth of hyphae from swollen mycelial fragments and by the re-establishment of permeability barriers resulting in the uptake of amino acids from the medium.     

Antibiotic production by Pseudomonas reptilivora as a phage conversion

The ability of Pseudomonas reptilivora to produce three broad-spectrum antimicrobial substances is easily lost when bacteria are subcultured. The study of the antibiotic production under defined culture conditions has shown that the biosynthesis of these substances depends upon the presence of a temperature-sensitive temperate phage. Antibiotic production is lost after phage induction.     

Antibiotic production by the cyanobacteriumNostoc muscorum

A broad spectrum antimicrobial antibiotic is produced byNostoc muscorum (Lancashire Polytechnic Culture Collection 23) during the post-exponential phase of growth. The antibiotic inhibits the growth of bacteria, notably multiple-resistantStaphylococcus aureus, and a biocide resistantPseudomonas aeruginosa: fungi such as the biodeteriogens,Cladosporium herbarum andHormoconis resinae and yeasts such asCandida albicans andC. pseudotropicalis. The antibiotic has an apparent molecular weight of 2000–3000 Daltons. Production appears to be dependent upon the limitation of one or more nutrients in the medium. author for correspondence     

Antibiotic production by bacterial biocontrol agents

Interest in biological control of plant pathogens has been stimulated in recent years by trends in agriculture towards greater sustainability and public concern about the use of hazardous pesticides. There is now unequivocal evidence that antibiotics play a key role in the suppression of various soilborne plant pathogens by antagonistic microorganisms. The significance of antibiotics in biocontrol, and more generally in microbial interactions, often has been questioned because of the indirect nature of the supporting evidence and the perceived constraints to antibiotic production in rhizosphere environments. Reporter gene systems and bio-analytical techniques have clearly demonstrated that antibiotics are produced in the spermosphere and rhizosphere of a variety of host plants. Several abiotic factors such as oxygen, temperature, specific carbon and nitrogen sources, and microelements have been identified to influence antibiotic production by bacteria biocontrol agents. Among the biotic factors that may play a determinative role in antibiotic production are the plant host, the pathogen, the indigenous microflora, and the cell density of the producing strain. This review presents recent advances in our understanding of antibiotic production by bacterial biocontrol agents and their role in microbial interactions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antibiotic production by a Roseobacter clade-affiliated species from the German Wadden Sea and its antagonistic effects on indigenous isolates

A strain affiliated with the Roseobacter clade and producing a new antibiotic named tropodithietic acid (L. Liang, Ph.D. thesis, University of G?ttingen, G?ttingen, Germany, 2003) was isolated from the German Wadden Sea. The compound showed strong inhibiting properties with respect to marine bacteria of various taxa and marine algae. Antibiotic production was found to occur during the complete growth phase. Strain mutants without antagonistic properties appeared several times spontaneously.     

Enhancement of Cephalosporin C production by cultivation of Cephalosporium acremonium M25 using a mixture of inocula

AIMS: To enhance the productivity of Cephalosporin C (CPC) by cultivation of Cephalosporium acremonium M25 using a mixture of inocula. METHODS AND RESULTS: Inoculum age was classified into three stages (early, intermediate and late) by image analysis. A mixture of inocula, according to the inoculum ages, was used for efficient production of CPC in the main culture. The most effective mixing ratio of inocula for CPC production in shake flasks was a 3 : 7 volume ratio of early- and late-stage inocula. This was also the case in a 1.5 l stirred-tank reactor. CPC productivity was enhanced by about 32% and 34% when using an inoculum mixture in the shake flask and 1.5 l stirred-tank reactor, respectively. CONCLUSION: The morphological characteristics of C. acremonium M25 in the seed culture were quite different according to inoculum age. The compromise of different ages of inoculum showed better production of CPC. Significance and Impact of the Study: The productivity of CPC was enhanced considerably when using mixed inocula. The results of this study can be applied to fungal cultures for efficient production of various metabolites.     

Cellulase production by a thermophilic clostridium species

Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 μm) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C₁ cellulase (degrades native cellulose) and C_x cellulase (β-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl₂ precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C_x cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature.     

Production of trypsin inhibitor by a Cephalosporium sp.

Conditions for the production of the trypsin inhibitor from Cephalosporium sp. KM 388 were investigated. Polypeptone-meat extract-glucose medium supported excellent production of the trypsin inhibitor. In this medium, polypeptone and meat extract were utilized both as carbon and nitrogen sources and as limiting substrates for the cell growth. Glucose was consumed during the stationary growth phase and prevented the disappearance of inhibitor activity. Cephalosporium sp. KM 388 grew at a rate of a first-order reaction for the cell concentrations. Trypsin inhibitor production paralleled cell growth. At 27 degrees C the maximum specific rates of growth and inhibitor production were 0.14 h-1 and 2.1 U of inhibitor/h per mg of cell, respectively. The production rate and the maximum yield of the inhibitor were increased 1.5- and 1.2-fold, respectively, when the initial pH 6.3 was maintained throughout the fermentation.     

Production of trypsin inhibitor by a Cephalosporium sp.

Conditions for the production of the trypsin inhibitor from Cephalosporium sp. KM 388 were investigated. Polypeptone-meat extract-glucose medium supported excellent production of the trypsin inhibitor. In this medium, polypeptone and meat extract were utilized both as carbon and nitrogen sources and as limiting substrates for the cell growth. Glucose was consumed during the stationary growth phase and prevented the disappearance of inhibitor activity. Cephalosporium sp. KM 388 grew at a rate of a first-order reaction for the cell concentrations. Trypsin inhibitor production paralleled cell growth. At 27 degrees C the maximum specific rates of growth and inhibitor production were 0.14 h-1 and 2.1 U of inhibitor/h per mg of cell, respectively. The production rate and the maximum yield of the inhibitor were increased 1.5- and 1.2-fold, respectively, when the initial pH 6.3 was maintained throughout the fermentation.     

Antibiotic production by Streptomyces aureofaciens using self-cycling fermentation

The self-cycling frementation (;rSCF) technique was applied to culture of Streptomyces aureofaciens. SCF is a method of continuous fermentation in which the metabolism of a microorganism is monitored by a measurement such as dissolved oxygen. These data are sent to a computer to allow it to control the system. Tetracycline production was observed only at exceedingly low iron concentrations in the growth medium. Repeatability of cycles was found to be dependent upon the presence of tetracycline in the fermentation broth as well as the strain of microorganism grown in the fermentor. Tetracycline was produced by an improved specific rate when compared to results in the literature for this organism grown using the batch method. (c) 1994 John Wiley & Sons, Inc.     

Xylanase active at high pH from an alkalotolerant Cephalosporium species

Summary An alkalotolerant Cephalosporium (NCL 87.11.9) strain capable of rapid growth and xylanase secretion over a wide pH range (pH 4–10) has been isolated from soil samples. When grown in shake flasks on a wheat bran, yeast extract medium for 96 h it produced 15 to 18 IU/ml. The novel feature of this study is that it is the first report of an extracellular fungal xylanase which is active and stable at high alkaline pH (8–9.5). The culture filtrate did not show any significant cellulase activity. Gel filtration studies indicated two peaks of xylanase activities corresponding to molecular weights of 70,000 and 30,000 in the proportion of 10:90.     

Bikaverin production by Fusarium species

Production of bikaverin has been examined in 130Fusarium isolates belonging to 21 species. The highest yield of bikaverin was produced on autoclaved rice — up to 2.5g/kg of dry culture. Bikaverin was produced by the following species:F verticillioides, F sacchari varsubglutinans, F proliferatum, F anthophilum, F oxysporum, F dlamini, F nygamai, F napiforme, andF solani. SpeciesF coeruleum, F poae, F sporotrichioides, F tricinctum, F chlamydosporum, F culmorum, F graminearum, F cerealis (F crookwellense), F avenaceum, F acuminatum, andF equiseti did not produce bikaverin. The production of bikaverin determines the colour of the mentionedFusarium species cultures on agar media and on rice. The pigment has indicator properties and changes colour from red in acidic solution to violet-blue in alkaline. The role it plays in fungus metabolism is not elucidated.     

Characteristics of Protease Production by Cephalosporium sp

We investigated protease formation by Cephalosporium sp. strain KM388, which produced trypsin inhibitor in the same cultures, in medium containing polypeptone, meat extract, and glucose (natural medium) and in medium containing NaNO₃, glucose, and yeast extract (semisynthetic medium). In natural medium, protease was secreted into the culture broth after cessation of growth caused by consumption of the polypeptone, the growth-limiting substrate. Enzyme formation in the stationary growth phase was due to de novo and so-called preferential synthesis, because cycloheximide immediately inhibited enzyme formation. In semisynthetic medium, protease was produced in parallel with mycelial growth, but production was repressed by the addition of polypeptone to the medium; protease production began after the added polypeptone was consumed. On the other hand, if glucose was eliminated from natural medium, the lag period of initiation of enzyme production was reduced until the late exponential phase. The addition of phosphate up to a concentration of 1.0% to natural medium also shortened the lag period and damped the pH change of the broth during cultivation.     

Identification and production of a rhamnolipidic biosurfactant by a Pseudomonas species

 A glycolipid-producing bacterium, Pseudomonas aeruginosa GL1, was isolated from the soil contaminated with polycyclic aromatic hydrocarbons (PAH) from a manufactured gas plant. The glycolipid produced was characterized in detail by chromatographic procedures as a mixture of four rhamnolipids, consisting of different associations of rhamnose and hydroxy fatty acids: the main component was monorhamnosyl di-3-hydroxydecanoic acid. The rhamnolipid composition presented marked analogies with a defined part of P. aeruginosa outer membrane lipopolysaccharides (lipopolysaccharide band A). Rhamnolipid production was stimulated under conditions of nitrogen limitation. Glycerol yielded higher productions than did hydrophobic carbon sources. Cell hydrophobicity decreased during growth on glycerol and on n-hexadecane whereas glycolipid production increased. P. aeruginosa GL1 was found to be unable to grow on a variety of 2, 3 and 4 cycle PAH. However, it was shown to persist after at least 12 subcultures in a bacterial population growing on a mixture of pure PAH, suggesting a physiological role for rhamnolipid as a means to enhance PAH availability in a mutualistic PAH-degrading bacterial community. Received: 4 July 1995/Received revision: 7 September 1995/Accepted: 13 September 1995     

Cellulase and beta-glucosidase production by a basidiomycete species.

The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose.     

Neuraminidase production by Bacteroides species

Abstract 128 strains of Bacteroides isolated from clinical specimens were surveyed for their ability to produce neuraminidase. All strains of Bacteroides fragilis and the B. fragilis group were neuraminidase-positive, as were strains of B. oralis and B. bivius . All strains of B. capillosus, B. ruminicola, B. disiens, B. multiacidus and B. uniformis did not produce a detectable neuraminidase. When human erythrocytes were exposed to cell extracts of neuraminidase-producing Bacteroides , and then tested with peanut ( Arachis hypogeae ) lectin, agglutination occurred. It was concluded that the production of neuraminidase by clinical isolates of Bacteroides may be associated with the pathophysiology of severe Bacteroides infections.