Role of Acetyl CoA: Deacetylcephalosporin C Acetyltransferase in Cephalosporin C Biosynthesis by Cephalosporium acremonium

Existence of an acetyltransferase, which catalizes acetylation of deacetylcephalosporin C to cephalosporin C, was demonstrated for the first time in cell-free extracts of Cephalosporium acremonium. The pH optimum of the enzyme appeared to be 7.0 to 7.5 and the enzyme required essentially Mg²⁺ as a cofactor for its reaction. The activity of this enzyme was not observed in the cell-free extracts of deacetylcephalosporin C-producing mutants Nos. 20, 29, 36 and 40, but was recovered in a revertant obtained from the mutant No. 40. These results indicate that deacetylcephalosporin C accumulation by these mutants was due to the lack of the acetyltransferase and made it reasonable that the terminal reaction of cephalosporin C biosynthesis in Cephalosporium acremonium proceeded by the catalytic action of acetyltransferase.     

Endogenous Inhibitors for Pollen Germination and Pollen Tube Elongation in Pinus densiflora SIEB,et Zucc.

Sulfur metabolism in Cephalosporium acremonium was investigated using a mutant, 8650⁺/ OAH^?/SeMe^R, which could not convert cysteine or inorganic sulfur to methionine. The production of cephalosporin by the mutant depended on the amount of S-sulfocysteine in a chemically defined medium supplemented with a low level of methionine sufficient to support optimal growth. S-Sulfocysteine was detected in an extract of cells grown in the presence of sodium thiosulfate and l-serine. Furthermore, an NADPH-linked reduction of S-sulfocysteine to cysteine was demonstrated in a cell-free extract. These facts suggest that S-sulfocysteine is a direct precursor in cysteine biosynthesis in C. acremonium and an alternative pathway involving the compound is one of the most important ones in cephalosporin C production by this fungus.     

Cephalosporium salvinae sp. nov. a pathogen of Salvinia rotundifolia willd

A new species ofCephalosporium is reported that is parasitic on the floating water fernSalvinia rotundifolia. In culture this fungus forms white, pulvinate colonies with a surface mat that may change to vinaceous-brown or violet with age. Small, ovate to spherical, hyaline conidia and spherical to elliptic chlamydospores are formed by this species. It represents a new member of theC. acremonium group. The pathogenic nature of the fungus has been confirmed.

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Zusammenfassung Eine neue Art vonCephalosporium ist beschrieben, die parasitisch an Wasserfarn,Salvinia rotundifolia, ist. In Kultur bildet dieser Pilz weiße, staubige Kolonien, die mit dem Alter braun oder violett werden. Diese Art bildet kleine ovale oder runde, hyaline Konidien und elliptische Chlamydosporen. Dieser Pilz ist ein neues Mitglied in derC. acremonium-Gruppe. Die pathogenetische Natur des Pilzes ist bestätigt worden.

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Based in part on a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, Atlanta University.

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Published as Research Publication No. 25 Atlanta University Center Science Research Institute.     

Revisione di alcuniCephalosporium isolati da casi patologici umani

Summary Eight species ofCephalosporium from man are revised, mostly on original strains.Cryptomyces pleomorpha Gruner is a typicalCephalosporium acremonium; in additionCryptomyces is a valid Phacidiaceous genus described more than a century ago. Of two human strains of the same species, one has been fully pathogenic for laboratory animals. Allantospora violacea Ambr.,A. onychophila Vuill. and an unnamed strain were revised. All are to be referred toCephalosporium (Allantospora)onychophilum (Vuill.) Coudert.Two strains ofAcremonium kiliense are typicalC. acremonium. C. serrae Maffei is a good species, but probably indistinguishable from the saprophyticC. subverticillatum Schulz et Sacc., reisolated in Italy in the year 1934. C. spinosum Negroni — probably indistinguishable fromC. cordoniformis Barbosa, both South American species — is a coremioidal form ofC. acremonium. C. rubrobrunneum (?) Benedek is a simpleC. acremonium, as well asC. stuehmeri Schmidt et van Beyma.In addition to the previously named species,C. pseudofermentatum Cif. (if really possess a yeast-like stage) andC. nigrum Kamb. (if this species is pertaining to the genusCephalosporium) are listed.

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Queste note furono redatte in gran parte tra il 1938 e il 1939; poi, per varie ragioni, rimasero inedite. Le ripubblichiamo con dati bibliografici aggiornati ed un maggiore materiale critico, anche in omaggio al comune amico, il compianto Prof.P. Redaelli.     

Stimulation of cell-free ring expansion of penicillin N by sonication and Triton X-100

Summary Cell-free protoplast lysates of Cephalosporium acremonium convert penicillin N to deacetoxycephalosporin C by ring expansion. Filtration eliminates 90% of their activity, indicating that the enzyme activity is particulate. The surfactant Triton X-100 or sonication of the lysate stimulates protoplast lysate activity. Active extracts could be produced by merely sonicating intact mycelia.     

Enhancement of growth and antibiotic titre inCephalosporium acremonium induced by sesame oil

The role of sesame oil as part of the carbon source on growth and cephalosporin C production byCephalosporium acremonium was studied in shake-flask fermentation. The growth and antibiotic production were maximum on the fifth and sixth day, respectively, irrespective of the presence of sesame oil. Sesame oil enhanced cephalosporin C production by 54%. Analysis of fatty acid profile indicated that C_18∶1, C_18∶2 and C_18∶3 are the major fatty acids inC. acremonium. The percentage of C_18∶2 was higher in the culture grown with sesame oil.     

Psoralea corylifolia extracts stimulate cholinergic-like psoralen receptors of tadpole-tail melanophores,leading to skin darkening

The present work was carried out to determine the effects of lyophilized seed extracts of Psoralea corylifolia along with pure psoralen, its active ingredient on the isolated tail-piece melanophores of Bufo melanostictus, a type of disguised smooth muscle cells, which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. In the present study, it was found that lyophilized extract of P. corylifolia and its active ingredient psoralen induced powerful, dose-dependent, physiologically significant melanin dispersal effects in the isolated tail melanophores of B. melanostictus, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of P. corylifolia and its active ingredient psoralen were highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of P. corylifolia and psoralen are mediated by cholino-muscarinic or cholino-psoralen like receptors having similar properties that need to be studied further.     

BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

Background  

Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas) in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration.     

Evolving enzyme technology for pharmaceutical applications: case studies

The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent. Received 07 March 1997/ Accepted in revised form 15 June 1997     

Modeling the effect of oxygen mass transfer on β-lactam antibiotic production by immobilizedCephalosporium acremonium

Summary The oxygen effect on the production of -lactam antibiotics was investigated whenCephalosporium acremonium was immobilized in calcium alginate. Theoretical effectiveness factors were calculated and compared with experimental ones. The approximate expressions of effectiveness factors reported in the literature were compared with the numerically obtained solutions to provide the best approximated expression for immobilizedC. acremonium cells. Yamane's approximate expression of effectiveness factor was best fit with the solutions numerically obtained in this investigation.     

Comparative lipidomic analysis of <Emphasis Type="Italic">Cephalosporium acremonium</Emphasis> insights into industrial and pilot fermentations

Cephalosporium acremonium has been widely applied in industrial cephalosporin C fermentation. However, little is known about the molecular basis of fermentation behavior of this strain. In this study, comparative lipidomic analysis using LC/ESI/MSⁿ technology was employed to investigate responses of Cephalosporium acremonium to multiple environment variations in realistic industrial cephalosporin C fermentation process and provide molecular basis for the discrepancies between industrial and pilot fermentations. Totally 77 phospholipids species were detected and 65 species were further quantified. Score plot revealed that phospholipids metabolism differed in industrial and pilot process. Loading pilot indicated that the main variables responsible for the discrimination of industrial and pilot process were phosphatidylinositols (PIs), phosphatidylserines (PSs) and phosphatic acids (PAs). Higher PIs content in industrial process indicated that cells were more vigorous in industrial process than those in pilot process. Larger increases of PSs, PAs and ratio of oleic acid to linoleic acid coincided well with the earlier and more thorough cellular morphological differentiation in industrial process. The synergetic reaction between cellular behavior and cells living environment led to titer discrepancies between industrial and pilot process. These findings provided lipidomic insights into industrial cephalosporin C production.     

Nitrofuran-Reducing Enzymes of Euglena*

SYNOPSIS. Euglena gracilis strain Z, green, dark-grown, and “bleached”with N-methyl-N-nitro-N-nitrosoguanidine, was found to contain 2 soluble enzymes which reduce nitrofurans. A small amount of activity was demonstrated also in a particulate fraction of sonic extracts, but none in isolated chloroplasts. The reduction of 5 nitrofurans, having widely differing bleaching activities, by each of the 2 enzymes was examined.     

In vitro evaluation of Colombian plant extracts against Black Sigatoka (Mycosphaerella fijiensis Morelet)

In this work, 90 dichloromethane and methanol extracts obtained from 45 plants collected at the Natural Reserve Bremen – La Popa (Colombia) and at the Natural Regional Park Ucumarí (NRPU, Colombia) belonging to five botanical families were evaluated at 1000 mg/l, for their in vitro fungicide activity through the ascospore germ tube elongation and the measurement of the mycelial radial growth of Mycosphaerella fijiensis assays. The methanol extracts from the species Lycianthes acutifolia (Solanaceae) and Piper pesaresanum (Piperaceae); as well as, the dichloromethane extracts from P. pesaresanum and those from the Lauraceae family named Nectandra acutifolia and Ocoteca paulii, all inhibited M. fijiensis ascospore germination in 100% in the germinative tube elongation assay. With regards to the effects of the plant extracts on mycelial radial growth, the methanol extracts from P. pesaresanum and the dichloromethane one from N. acutifolia both showed 100% inhibition in this bioassay. Additionally, from the phytochemical screening on the dichloromethane and methanol extracts it was found that compounds such as alkaloids, phenols and terpenes were present in most of the extracts evaluated and they might be the cause of the antifungal activities reported.     

Expression of a truncated ripening inhibitor (RIN) protein from tomato and production of an anti-RIN antibody

The tomato ripening mutant, ripening inhibitor (rin), whose fruits fails to ripen, has been identified and widely studied. The RIN gene has been cloned. Here we present the expression of a truncated form of the RIN protein from tomato and the preparation of a polyclonal antibody against it. The resulting antibody recognized the RIN of crude protein extracts from different tomato tissues. The protein level of RIN in tomato was detected with this antibody by western blot, which suggested the accumulation of RIN protein increased gradually during tomato fruit ripening. Hong-Liang Zhu and Ben-Zhong Zhu contributed equally to this work.     

Reconstitution of cyanobacterial photophosphorylation by a latent Ca2+-ATPase.

Photosynthetic membranes derived from sonic extracts of the cyanobacterium $\text{Spirulina}\text{platensis}$ contain a latent Ca⁺²-ATPase which is activated by exposure to trypsin. When sonic membranes are washed with ethylenediaminetetraacetic acid, the ATPase is removed from these membranes with an accompanying loss of photophosphorylation activity. The latent ATPase activity solubilized by washing has been partially purified, and addition of the enzyme to depleted membranes restores photophosphorylation activity to levels approaching 50% of the rates observed in unwashed membranes. These data indicate that this ATPase is the coupling factor responsible for photosynthetic energy transduction in $\text{Spirulina}\text{platensis}$.     

Occurrence of antibiotic activity in Conocephalum conicum,Mnium undulatum and Leptodictyum riparium (Bryophytes)

Abstract

In the present work the results on antibiotic activity of the extracts from the liverwort Conocephalum conicum and the mosses Minium undulatum and Leptodictyum riparium, tested against 8 bacterial strains pathogenic to man, are referred to. The extracts have been obtained, according the method suggested by McCleary et al. (1960), in either organic solvents or in water solution. The extracts have been proved active on both bacteria Gram + ve (G +) and Gram—ve (G—), in addition the extracts from Conocephalum conicum and Leptodictyum riparium (extracts in organic solvents in particular) were the most effective in the tests. The acetone extracts from Leptodictyum riparium showed the highest value of inhibition against Pseudomonas aeruginosa (bacterium responsible for serious opportunist infections). Our results on antibiotic activity of Bryophytes only partly agree with literature for either species showing activity or for the intensity of such activity. Among the three bryophytes employed Leptodictyum riparium showed the best inhibitory power against all bacteria tested. This result seems particularly interesting in relation to the possible competition between species occurring in the water where this moss preferentially grows. The phenomenon of antibiosis in the bryophytes is discussed also in relation to symbiotic or commensal relationships existing with soil bacteria and/or fungi.     

Iscephalosporium acremonium capable of producing maduromycosis?

Summary A case of mycetoma apparently due toCephalosporium acremonium is described. The position ofCephalosporium spp. as agents of maduromycosis is briefly considered.     

In vitro phosphorylation ofEscherichia coli isocitrate lyase

The in vitro phosphorylation of isocitrate lyase was demonstrated in partially purified sonic extracts ofEscherichia coli. Extracts were incubated with [gamma³²P]-ATP and subsequently analyzed by two-dimensional polyacrylamide gel electrophoresis. Isocitrate lyase was determined to be phosphorylated by autoradiography and Western blot analyses of the gels. Purified isocitrate lyase comigrates with the phosphorylated form of the enzyme; this suggests that the enzyme may become catalytically active concomitant with phosphorylation.     

Cloning and sequencing of the putative glyceraldehyde-3-phosphate dehydrogenase gene from Cephalosporium acremonium and its application to heterologous gene expression

A candidate for Cephalosporium acremonium glyceraldehyde-3-phosphate dehydrogenase gene (GAP) was cloned using the corresponding Saccharomyces cerevisiae gene as a probe. It encoded peptides showing marked similarity with those from S. cerevisiae and Aspergillus nidulans. A fused gene, GAP-HPT, consisting of the putative GAP promoter and the open reading frame of the bacterial hygromycin B phosphotransferase gene (HPT) was constructed, and it was successfully used to transform C. acremonium to a hygromycin B-resistant phenotype.     

Purification and partial characterization of a proteolytic enzyme from Cephalosporium acremonium (Corda)

A proteolytic enzyme was isolated fromCephalosporium acremonium (Corda). The enzyme had a molecular weight of 28,200 and a pH optimum of 8.4–9.2. Proteolytic activity was inhibited by diisopropylfluorophosphate and mercuric chloride. Of the substrates tested, casein was most rapidly hydrolyzed.