Árpád Csernetics

High-affinity iron permease (FTR1) gene sequence-based molecular identification of clinically important Zygomycetes

The clinical importance of zygomycosis, an emerging and frequently fatal mycotic disease, has increased during recent years. This report describes an identification method based on PCR amplification and sequencing of the high-affinity iron permease 1 gene (FTR1). Primers and amplification protocols were established and tested for the identification of Rhizopus oryzae, Rhizopus microsporus var. rhizopodiformis, R. microsporus var. oligosporus, Rhizopus schipperae, Rhizopus niveus and Rhizopus stolonifer. Rhizomucor and Syncephalastrum could be identified at the genus level. PCR-restriction fragment length polymorphism analysis of the amplified gene fragment using AluI digestion distinguished three subgroups among the R. oryzae isolates.

Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides

The zygomycete Mucor circinelloides accumulates β-carotene as the main carotenoid compound. In this study, the applicability of some early genes of the general isoprenoid pathway to improve the carotenoid production in this fungus was examined. The isopentenyl pyrophosphate isomerase gene (ipi) was cloned and used together with the genes encoding farnesyl pyrophosphate synthase (isoA) and geranylgeranyl pyrophosphate synthase (carG) in overexpression studies. Transformation experiments showed that the first bottleneck in the pathway, from the aspect of carotenoid production, is the step controlled by the carG gene, but overexpression of the ipi and isoA genes also contributes to the availability of the precursors. Transformations with these isoprenoid genes in combination with a bacterial β-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone.

Agrobacterium tumefaciens-mediated transformation of the zygomycete fungus Backusella lamprospora

The Agrobacterium ‐mediated transformation was adapted to Backusella lamprospora, a zygomycete fungus closely related to Mucor. The transforming plasmid contained the hygromycin B resistance (hph) and the green fluorescent protein (gfp) genes under the control of the regulator regions of the Mucor circinelloides gpd1 gene. The presence of the hph and gfp genes in the transformants was detected by PCR. The introduced genes could also be amplified directly from the spores of the transformants. The transformation efficiency was investigated by fluorescence microscopy of the transformed spores. A gradual decrease in the hygromycin B resistance was observed during several cultivation cycles: the growth of the transformants on the selection medium became slower, and the detection of the introduced gene became more difficult. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Genetic Transformation of Zygomycetes Fungi

Zygomycetes fungi, especially members of the order Mucorales and Mortierellales are important from various biological, biotechnological and medical aspects. They are used as model organisms to answer biological questions in the fields of sexual differentiation (Sutter, 1975; Kuzina and Cerda- Olmedo, 2006; Idnurm et al., 2008), morphological dimorphism (Lubbehusen et al., 2003; Iturriaga et al., 2005), biosynthesis of carotenoids (Iturriaga et al., 2000; Sanz et al., 2002; Kuzina et al, 2006; Almeida and Cerda-Olmedo, 2008) or the light induction of gene regulation and metabolite production (Velayos et al., 2003; Quiles-Rosillo et al., 2005; Idnurm et al., 2006). Phycomyces blakesleeanus, Mucor circinelloides, M. mucedo, Rhizopus oryzae and Absidia glauca are the best-studied species. Rhizopus, Mucor and Gilbertella species may also be important causing post-harvest losses in agricultural products, or as spoilage microorganisms of certain foods (Csernetics et al., 2005). Several species belonging to the genera Rhizopus, Absidia, Rhizomucor, Mucor, Apophysomyces, Saksanea, Cunninghamella, Cokeromyces and Syncephalastrum are known as causative agents of frequently fatal opportunistic fungal infections in immunocompromised patients called as zygomycoses (Ribes et al., 2000; Chayakulkeeree et al., 2006; Papp et al., 2008). High mortality rates, difficulties in the diagnosis and non-treatability with the most widely used antifungal drugs are characteristic features of such infections. Studies on the molecular and genetic background of the pathogenicity of these fungi started only a few years ago (Papp et al., 2008).

Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides

Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

Transcription of the three HMG-CoA reductase genes of Mucor circinelloides

BackgroundPrecursors of sterols, carotenoids, the prenyl groups of several proteins and other terpenoid compounds are synthesised via the acetate-mevalonate pathway. One of the key enzyme of this pathway is the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which catalyses the conversion of HMG-CoA to mevalonate. HMG-CoA reductase therefore affects many biological processes, such as morphogenesis, synthesis of different metabolites or adaptation to environmental changes. In this study, transcription of the three HMG-CoA reductase genes (designated as hmgR1, hmgR2 and hmgR3) of the β-carotene producing Mucor circinelloides has been analysed under various culturing conditions; effect of the elevation of their copy number on the carotenoid and ergosterol content as well as on the sensitivity to statins has also been examined.ResultsTranscripts of each gene were detected and their relative levels varied under the tested conditions. Transcripts of hmgR1 were detected only in the mycelium and its relative transcript level seems to be strongly controlled by the temperature and the oxygen level of the environment. Transcripts of hmgR2 and hmgR3 are already present in the germinating spores and the latter is also strongly regulated by oxygen. Overexpression of hmgR2 and hmgR3 by elevating their copy numbers increased the carotenoid content of the fungus and decreased their sensitivity to statins.ConclusionsThe three HMG-CoA reductase genes of M. circinelloides displayed different relative transcript levels under the tested conditions suggesting differences in their regulation. They seem to be especially involved in the adaptation to the changing oxygen tension and osmotic conditions of the environment as well as to statin treatment. Overexpression of hmgR2 and hmgR3 may be used to improve the carotenoid content.

Integration of a Bacterial β-Carotene Ketolase Gene into the Mucor circinelloides Genome by the Agrobacterium tumefaciens -Mediated Transformation Method

Plasmids introduced in Mucor circinelloides (and most transformable Mucorales) tend to replicate autonomously, and hardly ever integrate in the genome. This is critical if we want to express exogenous genes, because plasmids are easily lost during vegetative growth, and the ratio of plasmid molecules/nuclei is invariably low. Linearized molecules of DNA have been used to get their genomic integration but the transformation efficiency drops extremely. We have developed and highly optimized an efficient Agrobacterium-mediated transformation system for M. circinelloides to facilitate the integration of transforming DNA in the genome of the recipient strain that could also be used for other Mucorales.

Molecular studies on zygomycetes fungi causing opportunistic infections

Zygomycosis is a group of opportunistic fungal infections caused by filamentous fungi belonging in the class Zygomycetes. Incidence of such infections shows an increasing tendency as a result of the widespread use of immunosuppressive therapy, intensive cancer chemotherapeutic regimens and broad-spectrum antimicrobial agents. High mortality rates, difficulties in their diagnosis and an intrinsic resistance to the most widely used antifungal drugs are characteristic features of zygomycoses. All these facts indicate that the development of new strategies to prevent and treat zygomycosis is urgently needed. This review intends to summarize the current state of the different molecular genetic studies on clinically important zygomycetes with a special attention to the most recent efforts to develop new molecular identification methods and to determine the possible virulence factors. Questions of the DNA-based identification as well as of the genetic manipulation techniques applied in zygomycetes will also be discussed.

Improvement of Industrially Relevant Biological Activities in Mucoromycotina Fungi

Mucoromycotina contain several biotechnologically important fungi, primarily in the orders Mucorales and Mortierellales, which are considered as excellent producers of lipases and proteases, organic acids, alcohol, carotenoids and polyunsaturated fatty acids. Here, we overview gene expression and metabolic engineering studies performed to improve industrially relevant activities in different Mucoromycotina fungi in the past decade. Genetic transformation methods used to manipulate these fungi, especially Rhizopus oryzae, Mucor circinelloides and Mortierella alpina, and promoters used successfully to express genes in them will be reviewed. Concerning the improvement of production of hydrolytic enzymes special attention has been paid to the heterologous expression of lipases, which are among the best characterized enzymes in these fungi. Main achievements in the improvement of lactic acid and fumaric acid production by Rhizopus enzymes have also been summarized. Expression of endogenous and exogenous terpenoid and carotenoid biosynthetic genes to enhance and modify the carotene production of Mucor circinelloides has been detailed. Finally, homologous and heterologous expression of Mucoromycotina fatty acid desaturases and elongases in fungi and plants has been overviewed with special attention to the production of arachidonic acid and γ-linolenic acid.

South Indian Isolates of the Fusarium solani Species Complex From Clinical and Environmental Samples: Identification, Antifungal Susceptibilities, and Virulence

Members of the Fusarium solani species complex (FSSC) are the most frequently isolated fusaria from soil. Moreover, this complex solely affects more than 100 plant genera, and is also one of the major opportunistic human pathogenic filamentous fungi, being responsible for approximately two-third of fusariosis cases. Mycotic keratitis due to Fusarium species is among the leading causes of visual impairment and blindness in South India, but its management is still challenging due to the poor susceptibility of the isolates to conventional antifungal drugs. Aims of the present study were to isolate South Indian clinical and environmental FSSC strains and identify them to species level, to determine the actual trends in their susceptibilities to antifungal therapeutic drugs and to compare the virulence of clinical and environmental FSSC members. Based on the partial sequences of the translation elongation factor 1α gene, the majority of the isolates—both from keratomycosis and environment—were confirmed as F. falciforme, followed by F. keratoplasticum and F. solani sensu stricto. In vitro antifungal susceptibilities to commonly used azole, allylamine and polyene antifungals were determined by the CLSI M38-A2 broth microdilution method. The first generation triazoles, fluconazole and itraconazole proved to be ineffective against all isolates tested. This phenomenon has already been described before, as fusaria are intrinsically resistant to them. However, our results indicated that despite the intensive agricultural use of azole compounds, fusaria have not developed resistance against the imidazole class of antifungals. In order to compare the virulence of different FSSC species from clinical and environmental sources, a Drosophila melanogaster model was used. MyD88 mutant flies having impaired immune responses were highly susceptible to all the examined fusaria. In wild-type flies, one F. falciforme and two F. keratoplasticum strains also reduced the survival significantly. Pathogenicity seemed to be independent from the origin of the isolates.