<Emphasis Type="SmallCaps">l</Emphasis>-Arabinose pathway engineering for arabitol-free xylitol production in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Xylose reductase (XR) is a key enzyme in biological xylitol production, and most XRs have broad substrate specificities. During xylitol production from biomass hydrolysate, non-specific XRs can reduce l-arabinose, which is the second-most abundant hemicellulosic sugar, to the undesirable byproduct arabitol, which interferes with xylitol crystallization in downstream processing. To minimize the flux from l-arabinose to arabitol, the l-arabinose-preferring, endogenous XR was replaced by a d-xylose-preferring heterologous XR in Candida tropicalis. Then, Bacillus licheniformis araA and Escherichia coli araB and araD were codon-optimized and expressed functionally in C. tropicalis for the efficient assimilation of l-arabinose. During xylitol fermentation, the control strains BSXDH-3 and KNV converted 9.9 g l-arabinose l⁻¹ into 9.5 and 8.3 g arabitol l⁻¹, respectively, whereas the recombinant strain JY consumed 10.5 g l-arabinose l⁻¹ for cell growth without forming arabitol. Moreover, JY produced xylitol with 42 and 16% higher productivity than BSXDH-3 and KNV, respectively.     

Enhancement of xylitol production by attenuation of intracellular xylitol dehydrogenase activity in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

To construct Candida tropicalis strains that produce a high yield of xylitol with no requirement for co-substrates, we engineered the yeast with an attenuated xylitol dehydrogenase (XDH) and then assessed the efficiency of xylitol production The mutants, strains XDH-5 (with only one copy of the XDH gene), and ARSdR-16 (with a mutated XDH gene) showed 70 and 40% of wild type (WT) XDH activity, respectively. Conversions of xylose to xylitol by WT, XDH-5, and ARSdR-16 were 62, 64, and 75%, respectively, with productivities of 0.52, 0.54, and 0.62 g l⁻¹ h⁻¹, respectively. The ARSdR-16 mutant strain produced xylitol with high yield and high productivity in a simple process that required no co-substrates, such as glycerol. This strain represents a promising alternative for efficient and cost-effective xylitol production.     

A Fatal Case of <Emphasis Type="Italic">Candida auris</Emphasis> and <Emphasis Type="Italic">Candida tropicalis</Emphasis> Candidemia in Neutropenic Patient

We report a fatal case of Candida auris that was involved in mixed candidemia with Candida tropicalis, isolated from the blood of a neutropenic patient. Identification of both isolates was confirmed by amplification and sequencing of internal transcribed spacer and D1/D2 domain of large subunit in rRNA gene. Antifungal susceptibility test by E-test method revealed that C. auris was resistant to amphotericin B, anidulafungin, caspofungin, fluconazole, itraconazole and voriconazole. On the other hand, C. tropicalis was sensitive to all antifungal tested. The use of chromogenic agar as isolation media is vital in detecting mixed candidemia.     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L⁻¹, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L⁻¹, respectively, with an overall xylitol yield of 0.28 g g⁻¹. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.     

Transformation of a filamentous fungus<Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> using<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1×10⁶ conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.     

Symbiotic relationship between <Emphasis Type="Italic">Microbacterium</Emphasis> sp. SK0812 and <Emphasis Type="Italic">Candida tropicalis</Emphasis> SK090404

A bacterium growing inside yeast cytoplasm was observed by light microscope without staining. The bacterium was separately stained from yeast cell by a fluorescent dye, 4′,6-diamidino-2-phenylindole (DAPI). The bacterium actively moved inside yeast cytoplasm and propagated in company with the yeast growth. The bacterium was separated from the yeast cytoplasm by selective disruption of yeast cells and the yeast without the intracellular bacterium (YWOB) was obtained by selective inactivation of bacterial cells. The yeast and the intracellular bacterium were identified as Candida tropicalis and Microbacterium sp., respectively. The length of Microbacterium sp. and C. tropicalis measured with SEM image was smaller than 0.5 μm and was larger than 5 μm, respectively. The yeast with the intracellular bacterium (YWIB) grew in a starch-based medium but the YWOB was not C. tropicalis has neither extracellular nor intracellular saccharification enzyme. Glucose was produced from starch by the extracellular crude enzyme (culture fluid) of Microbacterium sp. YWIB produced significantly more ethanol from glucose than YWOB but did not from starch. Conclusively, C. tropicalis is thought to catabolize starch dependent upon Microbacterium sp. growing in its cytoplasm and furnish stable habitat for the Microbacterium sp.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

Ethanol production from hydrolysed agricultural wastes using mixed culture of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Acid, alkaline and enzymatic hydrolysis of agricultural crop wastes were compared for yields of total reducing sugars with the hydrolysates being evaluated for ethanol production using a mixed culture of Zymomonas mobilis and Candida tropicalis. Acid hydrolysis of fruit and vegetable residues gave 49–84 g reducing sugars l⁻¹ and 29–32 g ethanol l⁻¹ was then obtained. Alkaline hydrolysis did not give significant amount of reducing sugars. Enzymatic hydrolysis of fruit and vegetable residues yielded 36–123 g reducing sugars l⁻¹ and 11–54 g ethanol l⁻¹.     

Xylitol production by <Emphasis Type="Italic">Candida tropicalis</Emphasis> in a chemically defined medium

A chemically defined medium that included urea (5 g l(-1)) as a nitrogen source and various vitamins was substituted for a complex medium containing yeast extract (10 g l(-1)) in the production of xylitol by Candida tropicalis. In a fed-batch culture with the chemically defined medium, 237 g xylitol l(-1) was produced from 270 g xylose l(-1) after 120 h. The volumetric rate of xylitol production and the xylitol yield from xylose were 2 g l(-1) h(-1) and 89%, respectively. These values were about 5% lower and 4% higher, respectively, than those obtained using the complex medium. These results indicate that xylitol can be produced effectively in a chemically defined medium.     

Comparative Study of the Effects of Fluconazole and Voriconazole on <Emphasis Type="Italic">Candida glabrata</Emphasis>, <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida rugosa</Emphasis> Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC₅₀ of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.     

Paralysis and delayed Z-disc formation in the <Emphasis Type="Italic">Xenopus tropicalis</Emphasis><Emphasis Type="Italic">unc45b</Emphasis> mutant <Emphasis Type="Italic">dicky ticker</Emphasis>

Background  

The protein components of mature skeletal muscle have largely been characterized, but the mechanics and sequence of their assembly during normal development remain an active field of study. Chaperone proteins specific to sarcomeric myosins have been shown to be necessary in zebrafish and invertebrates for proper muscle assembly and function.     

Biodegradation of phenol and 4-chlorophenol by the yeast <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Biodegradation of phenol and 4-chlorophenol (4-cp) using a pure culture of Candida tropicalis was studied. The results showed that C. tropicalis could degrade 2,000 mg l⁻¹ phenol alone and 350 mg l⁻¹ 4-cp alone within 66 and 55 h, respectively. The capacity of the strain to degrade phenol was obviously higher than that to degrade 4-cp. In the dual-substrate system, 4-cp intensely inhibited phenol biodegradation. Phenol beyond 800 mg l⁻¹ could not be degraded in the presence of 350 mg l⁻¹ 4-cp. Comparatively, low-concentration phenol from 100 to 600 mg l⁻¹ supplied a sole carbon and energy source for C. tropicalis in the initial phase of biodegradation and accelerated the assimilation of 4-cp, which resulted in the fact that 4-cp biodegradation velocity was higher than that without phenol. And the capacity of C. tropicalis to degrade 4-cp was increased up to 420 mg l⁻¹ with the presence of 100–160 mg l⁻¹ phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and 4-cp as single and mixed substrates in batch cultures. The results illustrated that the models proposed adequately described the dynamic behaviors of biodegradation by C. tropicalis.     

<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.     

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.     

<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.     

Staged Reimplantation of a Total Hip Prosthesis After Co-infection with <Emphasis Type="Italic">Candida tropicalis</Emphasis> and <Emphasis Type="Italic">Staphylococcus haemolyticus</Emphasis>: A Case Report

Fungal prosthetic joint infection is a rare complication in total joint arthroplasty. There are no established guidelines for management of these infections. We present a case of a 53-year-old male with a hip joint prosthesis co-infected with Candida tropicalis and Staphylococcus haemolyticus. A two-stage exchange arthroplasty was performed. The patient underwent implant removal, debridement, irrigation with saline solution and application of cement spacer impregnated with vancomycin followed by aggressive antimicrobial treatment in first stage. Complete eradication of infection was demonstrated by negative culture of sonicated cement spacer fluid and negative 16S rRNA and 18S rRNA gene PCR of sonicate fluid, synovial fluid and periprosthetic tissue samples. He underwent second-stage revision hip arthroplasty after 9 months of the first stage. At the latest follow-up, there was no evidence of recurrence of infection. This case illustrates the utility of sonication of biomaterials and molecular techniques for microbiological confirmation of absence of infection in staged surgeries which is required for a successful outcome.     

Transformation of Industrialized Strain <Emphasis Type="Italic">Candida glycerinogenes</Emphasis> with Resistant Gene <Emphasis Type="Italic">zeocin</Emphasis> via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Candida glycerinogenes WL2002-5 has a modest sugar tolerance and an extremely high glycerol productivity. Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, into the nuclear genome of a wide variety of host cells. In this study, we constructed the plasmid pZR and transferred it into A. tumefaciens LBA4404 to form the strain LBA4404-ZR. LBA4404-ZR was cocultivated with C. glycerologenesis, and putative transformants were identified by selection for zeocin resistance. Polymerase chain reaction and Southern blot analysis confirmed that the gene zeocin was integrated into the genome of engineered C. glycerologenesis. Optimization of the transformation condition was performed in darkness at 25 degrees C on induction medium for 24 h by cocultivation of C. glycerinogenes and LBA4404-ZR with a cell ratio of 1:500-1000. The transformation efficiency reached 2 transformants per 10(4) C. glycerologenesis cells. Our results demonstrated that A. tumefaciens-mediated transformation can be used for C. glycerinogenes. This transformation system can provide the basis for research of C. glycerologenesis in the future.     

<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.     

Degradation of diesel oil by immobilized <Emphasis Type="Italic">Candida tropicalis</Emphasis> and biofilm formed on gravels

The performance of diesel oil degradation by Candida tropicalis immobilized on various conventional matrices (sodium alginate, carboxyl methyl cellulose, chitosan) and biowaste materials (wheat bran, sawdust, peanut hull powder) was investigated using the method of entrapment and physical adsorption. The yeast species immobilized in wheat bran showed enhanced efficiency in degrading diesel oil (98%) compared to free cells culture (80%) over a period of 7 days. Copious amount of exopolysaccharides were also produced in the presence of diesel oil. The biofilm forming ability of C. tropicalis on PVC strips was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy and atomic force microscopy. Yeast biofilm formed on gravels showed 97% degradation of diesel oil over a period of 10 days. The potential use of the biofilms for preparing trickling filters (gravel particles), for attenuating hydrocarbons in oily liquid wastes before their disposal in the open environment is suggested and discussed. This is the first successful attempt for ‘artificially’ establishing hydrocarbon degrading yeast biofilm on solid substrates.