Endophytic <Emphasis Type="Italic">Penicillium citrinum</Emphasis> Thom. from <Emphasis Type="Italic">Scoparia dulcis</Emphasis> Linn

Scoparia dulcis of Scrophulariaceae is an annual herb distributed through out the tropics. Penicillium citrinum was obtained from apparently healthy roots, stem, leaves and fruits of this plant. Callus and multiple shoots produced during micropropagation from various explants were also symptomless but showed occurrence of Penicillium citrinum when cultured in Murashige & Skoog liquid medium for the production of secondary metabolites.     

Transport of ergot alkaloids and quinocitrinins in the producing fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The decrease in the concentration of alkaloids in the culture liquid of Penicillium citrinum grown to the early stationary phase was found to be due to the uptake of quinocitrinins and ergot alkaloids by fungal cells. The ability of the fungal mycelium to take up autogenous quinocitrinins does not depend on the mycelium age, whereas its ability to take up ergot alkaloids is higher in the young than in the 12-day-old mycelium. The uptake of exogenously added ergot alkaloids by the fungal mycelium is accompanied by excretion of intracellular quinocitrinins. The addition of quinocitrinins to the medium was found to exert different effects in different growth stages. Namely, the uptake of exogenously added quinocitrinins by the actively growing young mycelium inhibits the excretion of ergot alkaloids, but the excretion of ergot alkaloids by the 12-day-old mycelium occurs throughout the cultivation period. The excretion of both ergot alkaloids and quinocitrinins does not require energy.     

Effect of quinocitrinines from the fungus Penicillium citrinum on the respiration of yeasts and bacteria

We studied the effect of quinocitrinines on the respiratory activity of yeasts (Yarrowia lipolytica) and bacteria (Arthrobacter globiformis). Quinocitrinines were shown to activate respiration of native cells in both types of organisms. Studies of yeast mitochondria showed that quinocitrinine exerts an uncoupling effect on oxidative phosphorylation, which activates the respiration, reduces the respiratory control, and decreases the ADP/O ratio. Experiments with intact mitochondria and native cells of Arthrobacter globiformis revealed that quinocitrinine decreases the membrane potential. The uncoupling effect likely constitutes a mechanism of the antibiotic activity of quinocitrinines.     

Effect of triton X-100 on compactin production from<Emphasis Type="Italic">Penicillium citrinum</Emphasis>

Glucose alone was found to be the most effective carbon source for producing compactin. An initial glucose concentration of 40 g/L gave the highest compactin concentration of 250 mg/L. Among the various nitrogen sources, when 5 g/L of pharmamedia and soybean meal as the sole nitrogen source were used, respectively, the compactin concentration was higher than 250 mg/L. Especially, in the case of the mixture of 6 g/L of pharmamedia and 8 g/L of soybean meal, the compactin concentration was 400 mg/L. To select the best surfactant for effective compactin production, various surfactants were investigated. When Triton X-100 was used, the maximum compactin concentration was 445 mg/L. With the initial concentration ranging from 1.5 to 2.0 g/L, the compactin concentration was the highest at 465–450 g/L. The cell concentration was similar to that of the control without the addition of Triton X-100. On the other hand, when the above 4.0 g/L of Triton X-100 were used, the cell concentration decreased. Using the based results the continuous fed-batch cultures by adding the Triton X-100 were carried out for 10 days in an air-lift bioreactor. When 1.5 g/L of Triton X-100 was added to the culture broth at 0, 4, and 8 days of culture, respectively, the compactin production was increased with the increase of culture time. The maximum compactin concentration after 10 days of culture was 1,200 mg/L, which was about 2.0-fold higher than that of the control without the addition of Triton X-100.     

Purification and characterization of pectin lyase secreted by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The molecular mass determined by SDS-PAGE was 31 kDa. The K _m and k _cat values were found to be 1 mg/ml and 76 sec⁻¹, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

A heterologous production of the <Emphasis Type="Italic">Trametes hirsuta</Emphasis> laccase in the fungus <Emphasis Type="Italic">Penicillium canescens</Emphasis>

A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta 072 accase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches 3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency of heterologous secretion. The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072.     

The influence of medium composition on alkaloid biosynthesis by <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The fungus P. citrinum produces secondary metabolites, clavinet ergot alkaloids (EA), and quinoline alkaloids (quinocitrinines, QA) in medium with various carbon and nitrogen sources and in the presence of iron, copper, and zinc additives. Mannitol and sucrose are most favorable for EA biosynthesis and mannitol is most favorable for QA. Maximum alkaloid production is observed on urea. Iron and copper additives in the medium containing zinc ions stimulated fungal growth but inhibited alkaloid biosynthesis. The production of these secondary metabolites does not depend on the physiological state of culture, probably due to the constitutive nature of the enzymes involved in biosynthesis of these substances.     

Carotenoids from <Emphasis Type="Italic">Rhodotorula</Emphasis> and <Emphasis Type="Italic">Phaffia</Emphasis>: yeasts of biotechnological importance

Carotenoids represent a group of valuable molecules for the pharmaceutical, chemical, food and feed industries, not only because they can act as vitamin A precursors, but also for their coloring, antioxidant and possible tumor-inhibiting activity. Animals cannot synthesize carotenoids, and these pigments must therefore be added to the feeds of farmed species. The synthesis of different natural commercially important carotenoids (β-carotene, torulene, torularhodin and astaxanthin) by several yeast species belonging to the genera Rhodotorula and Phaffia has led to consider these microorganisms as a potential pigment sources. In this review, we discuss the biosynthesis, factors affecting carotenogenesis in Rhodotorula and Phaffia strains, strategies for improving the production properties of the strains and directions for potential utility of carotenoid-synthesizing yeast as a alternative source of natural carotenoid pigments.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Novel cellulases from an extremophilic filamentous fungi <Emphasis Type="Italic">Penicillium citrinum</Emphasis>: production and characterization

The enzymatic hydrolysis of cellulose has potential economical and environment-friendly applications. Therefore, discovery of new extremophilic cellulases is essential to meet the requirements of industry. Penicillium citrinum (MTCC 6489) that was previously isolated from soil in our laboratory, produced alkali tolerant and thermostable cellulases. Endoglucanase and filter paper activity hydrolase (FPAse) production of P. citrinum were studied using wheat bran substrate in solid state and submerged culture. Zymogram analysis of endoglucanase revealed the presence of two isoforms differing in molecular weight. One of them was 90 kDa and other one was 38 kDa. Partially purified endoglucanase showed two different peaks at pH 5.5 and 8.0, respectively, in its pH optima curve. But FPase showed only one peak (at pH 6.5) in its pH optima curve. Cellulase of P. citrinum is thermostable in nature. The present work reports for the first time, the alkali stable cellulase from alkali tolerant fungus Penicillium citrinum. Thermostable endoglucanase from P. citrinum may have potential effectiveness as additives to laundry detergents.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Screening and molecular identification of lactic acid bacteria from <Emphasis Type="Italic">gari</Emphasis> and <Emphasis Type="Italic">fufu</Emphasis> and <Emphasis Type="Italic">gari</Emphasis> effluents

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

<Emphasis Type="Italic">Sporothrix inflata</Emphasis>, a root-inhabiting fungus of <Emphasis Type="Italic">Quercus robur</Emphasis> and <Emphasis Type="Italic">Q. petraea</Emphasis>

The anamorphic fungus Sporothrix inflata, known as a soil-borne fungus with worldwide distribution, was isolated for the first time from the cortex and central cylinder of living and dead roots of healthy and diseased oak trees (Quercus robur and Q. petraea). Isolation frequencies of S. inflata from oak roots varied according to the health status of trees, oak species, study sites, soil depth and root diameter. Colony morphology and growth rate of isolates are influenced by colony age and type of culture medium.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Continuous production of neo-fructooligosaccharides by immobilization of whole cells of <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

To produce neo-fructooligosaccharides (neo-FOSs) in a 500ml continuous packed-bed reactor using whole cell immobilization of Penicillium citrinum KCCM 11663, the optimum reaction conditions were 50 °C, pH 6 with 600 g sucrose l^-1 being fed as substrate at 1.3 ml min^-1 . Under these conditions, the maximum neo-FOSs production was 49 g l^-1. In a packed-bed reactor, continuous production of neo-FOSs was possible for 50 d indicating a potential for industrial production. Revisions requested 6 September 2004/14 October 2004; Revisions received 7 October 2004/29 November 2004