This work was aimed at identifying and at characterizing new Pleurotus ostreatus laccases, in order to individuate the most suitable biocatalysts for specific applications. The existence of a laccase gene clustering was demonstrated in this basidiomycete fungus, and three new laccase genes were cloned, taking advantage of their closely related spatial organization on the fungus genome. cDNAs coding for two of the new laccases were isolated and expressed in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, in order to optimize their production and to characterize the recombinant proteins. Analysis of the P. ostreatus laccase gene family allowed the identification of a “laccase subfamily” consisting of three genes. A peculiar intron–exon structure was revealed for the gene of one of the new laccases, along with a high instability of the recombinant enzyme due to lability of its copper ligand. This study allowed enlarging the assortment of P. ostreatus laccases and increasing knowledge to improve laccase production.

Carotenoids have been identified in the fungus Podospora anserina and a parallel pathway to neurosporene and β-carotene was established. Three genes for the β-carotene branch have been cloned and their function elucidated. They correspond to the al-1, al-2 and al-3 genes from Neurospora crassa. They were individually and in combinations over-expressed in P. anserina in order to modify the carotenoid composition qualitatively and quantitatively. In the resulting transformants, carotenoid synthesis was up to eightfold increased and several intermediates of the pathway together with special cyclic carotenoids, β-zeacarotene and 7,8-dihydro-β-carotene, accumulated. All transformants with an over-expressed al-2 gene (encoding a phytoene synthase and a lycopene cyclase) displayed up to 31% prolonged life span.

We compared the gene expression patterns of Lentinula edodes fresh fruiting bodies and fruiting bodies 3 days after harvest, by suppression subtractive hybridization, to characterize the physiologic changes that occur after harvest, such as gill browning and cell wall lysis of the fruiting body, which are responsible for the loss of food quality and value. We found increase of transcription levels of several enzyme encoding genes, such as, two phenol oxidases encoding genes (tyr tyrosinase, lcc4 laccase), and several cell wall degradation-related enzyme-encoding genes, such as mixed-linked glucanase (mlg1), chitinases (chi1, chi2), chitin deacetylase (chd1), and chitosanase (cho1), after harvesting. We isolated a putative transcription factor-encoding gene (L. edodes exp1) with high similarity to exp1 from Coprinopsis cinerea, which is involved in autolysis of the cap during spore diffusion. Transcription of L. edodes exp1 increased post-harvest, which suggests that its target genes are up-regulated after harvesting. These enzymes and the transcription factor may be involved in L. edodes fruiting body senescence.

The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization.

The yeast Pichia ciferrii produces large quantities of the sphingoid base tetraacetyl phytosphingosine (TAPS) and is an interesting platform organism for the biotechnological production of sphingolipids and ceramides. Ceramides have attracted great attention as a specialty ingredient for moisture retention and protection of the skin in the cosmetics industry. First attempts have been started to metabolically engineer P. ciferrii for improved production of TAPS and other sphingoid bases. However, rational metabolic engineering of P. ciferrii is difficult due to a low gene targeting efficiency. In eukaryotes, two major pathways coexist, which are responsible for genomic DNA integration, homologous recombination (HR) and non-homologous end joining (NHEJ). Integration via HR is targeted, while NHEJ involves ectopic (non-targeted) integration depending on a ligation step mediated by DNA ligase IV (Lig4). Here, we demonstrate a dramatical increase in gene targeting efficiency in a P. ciferriilig4 knockout strain, deficient in NHEJ. Furthermore, a quick and easy to use freeze–thaw method was developed to transform P. ciferrii with high efficiency. Owing to the ability of targeting genomic DNA integration our results pave the way for further genetic and metabolic engineering approaches with P. ciferrii by means of knocking out or overexpressing predestinated genes.

This study focuses on the cloning and characterization of the major nitrogen regulator element from the ectomycorrhizal fungus Tuber borchii, TbNre1. Sequence analysis of the predicted protein and complementation experiments in Neurospora crassa demonstrated that the cloned gene is orthologous to areA/nit-2 gene. Transcriptional expression investigations by real-time RT-PCR showed TbNre1 up-regulation in the presence of nitrate or in the absence of nitrogen during free-living mycelium growth. On the contrary, TbNre1 mRNA levels remained at basal values in the presence of preferred nitrogen sources like ammonium and glutamine. Furthermore, TbNre1 mRNA was found to be up-regulated during T. borchii and T. platyphyllos interaction. All these data suggest that the regulatory protein TBNRE1 could play a major role in regulating N metabolism genes of T. borchii in the free living mycelium and in T. borchii–T. platyphyllos interaction. Finally, the possible role of the transcription factor TBNRE1 in the induction of proteases and virulence-like genes, necessary in ectomycorrhizal establishment, was also discussed.

spl1-1 was originally identified as a spontaneous mutation genetically interacting with sep1-1 and cdc4-8 in producing multinucleate syncytia. This study shows that it is allelic with the proline-tRNA^CGG gene SPATRNAPRO.02. Its nucleotide sequence contains a C→T substitution in the region corresponding to the B-box of the putative intragenic promoter and the TψC loop of the mature tRNA. The substitution drastically reduces the transcription efficiency of the gene and pleiotropically affects numerous cellular processes. spl1-1 cells are temperature sensitive, osmosensitive, bend at higher temperatures, have extended G2 phase and are defective in cell separation (septum cleavage). The proline-tRNA^TGG gene SPATRNAPRO.01 can partially suppress the spl1-1 mutation when introduced into the cells on a multicopy plasmid. The effect of a mutation in a tRNA gene on cell separation brings a new element into the complexity of the regulation of cell division and its co-ordination with other cellular processes in Schizosaccharomyces pombe.

The yeast Candida parapsilosis is an opportunistic human pathogen frequently associated with nosocomial infections in neonates and patients with diminished immunity. A growing number of studies powered by recent advances in molecular genetics and genomics provide a background for uncovering the molecular basis of its virulence that suggests promising avenues for therapeutic intervention against this pathogen. Importantly, these studies also revealed several unique genetic and physiological features absent in model organisms, such as baker’s and fission yeasts. Hence, besides the clinical impact, C. parapsilosis represents an interesting non-conventional model suitable for investigations of several fundamental biological phenomena in cellular physiology, morphogenesis, and genome maintenance. In this study, we provide a concise review on C. parapsilosis biology and highlight its interesting biological features. In addition, we summarize approaches for genetic manipulation, which have enhanced research on this species by overcoming limitations of conventional genetic analysis caused primarily by an apparent absence of a sexual cycle and the diploid state of its genome.

In this study we provide general information on the little studied eukaryotic ribosomal protein rpL15. Saccharomyces cerevisiae has two genes, YRPL15A and YRPL15B that could potentially code for yeast rpL15 (YrpL15). YRPL15A is essential while YRPL15B is dispensable. However, a plasmid-borne copy of the YRPL15B gene, controlled by the GAL1 promoter or by the promoter controlling expression of the YRPL15A gene, can functionally complement YrpL15A in yeast cells, while the same gene controlled by the authentic promoter is inactive. Analysis of the levels of YrpL15B-mRNA in yeast cells shows that the YRPL15B gene is inactive in transcription. The function of YrpL15A is highly resilient to single and multiple amino acid substitutions. In addition, minor deletions from both the N- and C-terminal ends of YrpL15A has no effect on protein function, while addition of a C-terminal tag that could be used for detection of plasmid-encoded YrpL15A is detrimental to protein function. YrpL15A could also be replaced by the homologous protein from Arabidopsis thaliana despite almost 30% differences in the amino acid sequence, while the more closely related protein from Schizosaccharomyces pombe was inactive. The lack of function was not caused by a failure of the protein to enter the yeast nucleus.

Chitinases have been implicated in fungal cell wall remodeling and play a role in exogenous chitin degradation for nutrition and competition. Due to the diversity of these enzymes, assigning particular functions to each chitinase is still ongoing. The entomopathogenic fungus Metarhizium anisopliae produces several chitinases, and here, we evaluate whether endochitinase CHI2 is involved in the pathogenicity of this fungus. We constructed strains either overexpressing or lacking the CHI2 chitinase. These constructs were validated by Southern, Northern and Western blot analysis, and chitinase production. To access the effects of CHI2 chitinase in virulence, the cotton stainer bug Dysdercus peruvianus was used as a host. CHI2 overexpression constructs showed higher efficiency in host killing suggesting that the production of this chitinase by a constitutive promoter reduces the time necessary to kill the insect. More significantly, the knock out constructs showed decreased virulence to the insects as compared to the wild type strain. The lack of this single CHI2 chitinase diminished fungal infection efficiency, but not any other detectable trait, showing that the M. anisopliae family 18, subgroup B endochitinase CHI2 plays a role in insect infection.