Production of pigment-free pullulan by swollen cell in <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> NG which cell differentiation was affected by pH and nutrition

A black yeast strain “NG” was isolated from strawberry fruit and identified as Aureobasidium pullulans. Strain NG displayed yeast-like cell (YL), swollen cell (SC), septate swollen cell (SSC), meristematic structure (MS), and chlamydospore (CH) morphologies. pH was the key factor regulating cell morphogenesis of strain NG. Differentiation of YL controlled by extracellular pH had no relationship with nutrition level. YL was maintained at pH >6.0, but was transformed into SC at pH ∼4.5. SC, a stable cell type of A. pullulans, could bud, septate, or transform into MS or CH, in response to nutrition level and low pH. SC produced swollen cell blastospores (SCB) at pH 2.1 with abundant nutrition, and could transform into MS at lower pH (1.5). SC was induced to form CH by low level nutrition and pH <3, and this transition was suppressed by adjusting pH to ∼4.5. Crude polysaccharides without pigment (melanin) were produced by SC of strain NG. Pullulan content of the polysaccharides was very high (98.37%). Fourier-transform infrared spectroscopy confirmed that chemical structures of the polysaccharides and standard pullulan were identical. Swollen cells produced 2.08 mg/ml non-pigmented polysaccharides at 96 h in YPD medium. Controlling pH of fermentation is an effective and convenient method to harvest SC for melanin-free pullulan production.     

Characterization of<Emphasis Type="Italic"> Aureobasidium pullulans</Emphasis> isolated from airborne spores in Thailand

Isolates from air in several locations in Thailand were identified as Aureobasidium pullulans PR with dark pigmentation (Loei province), A. pullulans SU with an unusual conidial apparatus (Chiangmai province), and A. pullulans CU with burgundy-red pigmentation (from a shady area in Bangkok). The internal transcribed spacer sequences of the rDNA of A. pullulans SU and A. pullulans CU confirmed that they were A. pullulans. Both A. pullulans CU and A. pullulans PR preferred 30 °C and pH 7.5 for exopolysaccharide (EPS) production, while A. pullulans SU preferred 25 °C and pH 6.5. All three isolates preferred glucose over sucrose and (NH₄)₂SO4 over peptone for EPS production. Under optimal conditions, A. pullulans PR produced EPS yields of up to 0.225 g g⁻¹, followed by A. pullulans CU (0.185 g g⁻¹) and A. pullulans SU (0.158 g g⁻¹). Amylase activities were detected during the course of EPS production but gradually decreased as the EPS yields increased. IR spectra suggest that the EPS from these isolates was pullulan. EPS from the three isolates were partially sensitive to pullulanase. Electronic Publication     

Production,formulation and antagonistic activity of the biocontrol like-yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> against <Emphasis Type="Italic">Penicillium expansum</Emphasis>

Aureobasidium pullulans (de Bary) Arnaud (Ach 1-1) was grown in a glucose fed-batch fermentor to 106 g dry wt l⁻¹ in 48 h. The cells were dried in a fluidized bed dryer with a final viability of 62%. After 7 months at 4°C, the viability was 28% of the initial value (= 2.3 × 10¹⁰ c.f.u. g⁻¹ dry matter). A protection level of 89% was achieved with the biomass preparation at 1 × 10⁸ c.f.u. ml⁻¹ after 28 and 7 days for apples stored respectively at 5 and 25°C against Penicillium expansum. Our process is suitable to produce large quantities of the strain Ach 1-1 as biological control agent for apple preservation.     

Bioproducts from <Emphasis Type="Italic">Aureobasidium pullulans,</Emphasis> a biotechnologically important yeast

It has been well documented that Aureobasidium pullulans is widely distributed in different environments. Different strains of A. pullulans can produce amylase, proteinase, lipase, cellulase, xylanase, mannanase, transferases, pullulan, siderophore, and single-cell protein, and the genes encoding proteinase, lipase, cellulase, xylanase, and siderophore have been cloned and characterized. Therefore, like Aspergillus spp., it is a biotechnologically important yeast that can be used in different fields. So it is very important to sequence the whole genomic DNA of the yeast cells in order to find new more bioproducts and novel genes from this yeast.     

Cloning,characterization and expression of the extracellular lipase gene from <Emphasis Type="Italic">Aureobasidium </Emphasis><Emphasis Type="Italic">pullulans</Emphasis> HN2-3 isolated from sea saltern

The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.     

Pullulan production by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> cells immobilized in chitosan beads

Fungal cells of Aureobasidium pullulans ATCC 201253 were immobilized by entrapment in chitosan beads, and the immobilized cells were investigated for their ability to produce the polysaccharide pullulan using batch fermentation. The 1% chitosan-entrapped fungal cells were capable of producing pullulan for two cycles of 168 h using corn syrup as a carbon source. Pullulan production by the immobilized cells increased by 1.6-fold during the second production cycle (5.0 g/l) relative to the first production cycle (3.1 g/l) with the difference in production being statistically significant after 168 h. The productivity of the immobilized cells increased during the second production cycle while its pullulan content decreased. The level of cell leakage from the support remained unchanged for both production cycles.     

Thailand habitats as sources of pullulan-producing strains of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Summary A variety of habitats were sampled for the presence of Aureobasidium black yeasts with the attempt to find pullulan-producing strains. Habitats included leaves of mango (Mangifera indica Linn.), tamarind (Tamarindus indica Linn.), asoka (Saraca indica Linn.) and latex-painted and bathroom cement-wall surfaces. Parameters for the identification of the isolates included morphology, nutritional parameters, exopolysaccharide (EPS) production, and rDNA internal transcribed spacer (ITS) sequencing. All isolates of black yeasts were polymorphic with blastospores, hyphae, and chlamydospores. ITS analyses showed strong correlation with the GenBankA. pullulanssequences, with alignment using BLAST yielding greater than 95% similarity. All five isolates tested produced pullulan as deduced from infrared spectra and sensitivity to pullulanase. None produced aubasidan as evidenced from their IR spectra. The current studies support the notion that the hot, humid environments facilitate the development of A. pullulansand its tropical variants in diverse phylloplane and walls habitats, and merit support for further isolation and characterization of these black yeasts as a source of unique pullulan-producing strains.     

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.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Cloning,Characterization, and Expression of the Gene Encoding Alkaline Protease in the Marine Yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> 10

The alkaline protease structural gene (ALP1 gene) was isolated from both the genomic DNA and cDNA of Aureobasidium pullulans 10 by inverse PCR and RT-PCR. An open reading frame of 1248 bp encoding a 415 amino-acid protein with calculated molecular weight of 42.9 kDa was characterized. The gene contained two introns, which had 54 bp and 50 bp, respectively. The promoter of ALP1 gene was located from -62 to -112 and had two CCAAT boxes and one TATA box. The terminator of ALP1gene contained the sequence with a hairpin structure (AAAAAGTT TGGTTTTT). The protein sequence deduced from ALP1 gene exhibited 55.24%, 50.35%, and 31.68% identity with alkaline proteases from Aspergillus fumigatus, Acremonium chrysogenum, and Yarrowia lipolytica, respectively. The protein was found to have the conserved serine active site and histidine active site of serine proteases in the subtilisin family. The recombinant A. pullulans alkaline protease produced in Y. lipolytica formed clear zones on the double plates with 2% casein and alkaline protease activity in the supernatant of the recombinant Y. lipolytica culture was detected, suggesting that the cloned ALP1 gene is expressed in Y. lipolytica and the expressed alkaline protease is secreted into the medium.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Study of tauopathies by comparing <Emphasis Type="Italic">Drosophila</Emphasis> and human <Emphasis Type="Italic">tau</Emphasis> in <Emphasis Type="Italic">Drosophila</Emphasis>

The microtubule-binding protein tau has been investigated for its contribution to various neurodegenerative disorders. However, the findings from transgenic studies, using the same tau transgene, vary widely among different laboratories. Here, we have investigated the potential mechanisms underlying tauopathies by comparing Drosophila (d-tau) and human (h-tau) tau in a Drosophila model. Overexpression of a single copy of either tau isoform in the retina results in a similar rough eye phenotype. However, co-expression of Par-1 with d-tau leads to lethality, whereas co-expression of Par-1 with h-tau has little effect on the rough eye phenotype. We have found analogous results by comparing larval proteomes. Through genetic screening and proteomic analysis, we have identified some important potential modifiers and tau-associated proteins. These results suggest that the two tau genes differ significantly. This comparison between species-specific isoforms may help to clarify whether the homologous tau genes are conserved. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by the National Science Foundation of China (30270341; 30630028), the Multidisciplinary Program (Brain and Mind) of the Chinese Academy of Sciences, the Major State Basic Research Program (“973 program”; G2000077800; G2006CB806600; 2006CB911003), the Precedent Project of Important Intersectional Disciplines in the Knowledge Innovation Engineering of the Chinese Academy of Sciences (KJCX1-09-03).     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

Phenotypic Switching of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis><Emphasis Type="Italic">gattii</Emphasis>

Microorganisms that live in fluctuating environments must constantly adapt their behavior to survive. The host constitutes an important microenvironment in opportunistic and primary fungal pathogens like Cryptococcus neoformans (C. neoformans) and Cryptococcus gattii (C. gattii). In clonal populations, adaptation may be achieved through the generation of diversity. For fungi phenotype switching constitutes a mechanism that allows them to change rapidly. Both C. neoformans and C. gattii undergo phenotypic switching, which allows them to be successful pathogens and cause persistent disease. Similar to other encapsulated microbes that exhibit phenotypic variation, phenotypic switching in Cryptococcus changes the polysaccharide capsule. Most importantly, in animal models phenotypic switching affects virulence and can change the outcome of infection. Virulence changes because C. neoformans and C. gattii switch variants elicit different inflammatory responses in the host. This altered host response can also affect the response to antifungal therapy and in some cases may even promote the selection of switch variants. This review highlights the similarity and differences between phenotypic switching in C. neoformans and C. gattii, the two dominant species that cause cryptococcosis in humans.