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The gray mold fungus Botrytis cinerea features a wide host range and causes severe economic losses, making it an important object for molecular research. Thus far, genetic modification of the fungus mainly is relied on two selection systems (nourseothricin and hygromycin), while other selection systems hold significant disadvantages. To broaden the spectrum of available molecular tools, a new selection system based on the cheap and widely used fungicide fenhexamid (hydroxyanilide group) was established. Fenhexamid specifically targets the 3-ketoreductase ERG27 from the ergosterol biosynthesis pathway. We generated a set of expression vectors suitable for deletion or expression of genes of interest (GOIs) in B. cinerea based on fenhexamid-insensitive ERG27 variants. Expression of BcERG27F412I and Fusarium fujikuroi ERG27 in the sensitive B. cinerea strain B05.10 causes resistance towards fenhexamid (fenR) and allows for the selection of transformants and their genetic purification. A modified split-marker approach facilitates the site-specific integration and expression of GOIs at the bcerg27 locus. No undesired secondary phenotypes regarding virulence, stress responses, the formation of reproductive structures or conidial germination were observed in strains expressing fenhexamid-insensitive ERG27 variants. Thus, the fenR system represents a third reliable selection system for genetic modifications of fenhexamid-sensitive B. cinerea strains.

The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains. Here, we present a protocol for single-step marker switching by lithium acetate transformation in fission yeast, Schizosaccharomyces pombe. In the following we describe how to swap the ura4+ marker to a kanMX6, natMX4, or hphMX4 marker, which provide resistance against the antibiotics G418, nourseothricin (clonNAT) or hygromycin B, respectively. We also detail how to exchange any of the MX markers for nutritional markers, such as arg3+, his3+, leu1+ and ura4+ .

Clinical isolates are prototrophic and hence are not amenable to genetic manipulation using nutritional markers. Here we describe a new set of plasmids carrying the NAT1 (nourseothricin) drug resistance marker (Shen et al., ), which can be used both in clinical isolates and in laboratory strains. We constructed novel plasmids containing HA-NAT1 or MYC-NAT1 cassettes to facilitate PCR-mediated construction of strains with C-terminal epitope-tagged proteins and a NAT1-pMet3-GFP plasmid to enable conditional expression of proteins with or without the green fluorescent protein fused at the N-terminus. Furthermore, for proteins that require both the endogenous N- and C-termini for function, we have constructed a GF-NAT1-FP cassette carrying truncated alleles that facilitate insertion of an intact, single copy of GFP internal to the coding sequence. In addition, GFP-NAT1, RFP-NAT1 and M-Cherry-NAT1 plasmids were constructed, expressing two differently labelled gene products for the study of protein co-expression and co-localization in vivo. Together, these vectors provide a useful set of genetic tools for studying diverse aspects of gene function in both clinical and laboratory strains of C. albicans.

We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

In recent years a number of molecular tools have been reported for use in the human fungal pathogen Candida albicans, including PCR-mediated approaches for gene disruption, conditional expression and epitope tagging. Traditionally these methods have utilized auxotrophic markers; however, the availability of auxotrophic markers can be limiting and in some instances their use may also impact on the interpretation of results. As a result, the use of positive selection markers has now become more commonplace. Here we report the development and validation of a set of cassettes for PCR-mediated gene tagging and overexpression studies utilizing the nourseothricin resistance (CaNAT1) positive selection marker. In particular we have produced cassettes containing yeast-enhanced GFP, YFP, CFP, RFP and a combined V5-6xHis epitope tag. The cassettes are engineered for use in PCR-mediated gene tagging strategies where insertion is targeted to the 3' end of the gene of interest. In addition, to facilitate protein functional analysis and genetic suppression studies through the use of overexpression, we have also constructed a promoter replacement cassette containing the ENO1 promoter which is known to be expressed at a high level. These cassettes expand on the range of molecular tools available for working with C. albicans and may also be used in other Candida species that display sensitivity to nourseothricin.

Plants use multiple defence mechanisms comprising both constitutive and inducible barriers to prevent entering of phytopathogenic micro-organisms. In many plant species one of the most efficient responses to combat attacking microbes is the rapid synthesis of antimicrobial low molecular weight phytoalexins, for example, resveratrol, 3,5,4'-trihydroxystilbene (1). Resveratrol and its natural derivatives, however, display only moderate antimicrobial effects. Nevertheless, resveratrol may be a useful lead structure for the chemical synthesis of antimicrobials. In this study, several series of stilbenes have been synthesized, starting from the aldehydes using Wittig reactions to access the corresponding styrenes that were subjected to Mizoroki-Heck reactions to yield the stilbenes in good yields. The stilbenes were tested in an agar diffusion assay against several bacteria and fungi. For some of these compounds the inhibiting zones for bacteria and fungi were comparable with those of the antibiotics tetracycline, streptomycin, ampicillin, or kanamycin, directed against prokaryotes, and nourseothricin or hygromycin controlling fungi, respectively.

Yeasts associate with numerous insects, and they can assist the metabolic processes within their hosts. Two distinct yeasts were identified by PCR within the planthopper Perkinsiella saccharicida, the vector of Fiji disease virus to sugarcane. The utility of both microbes for potential paratransgenic approaches to control Fiji leaf gall (FLG) was assessed. Phylogenetic analysis showed one of the microbes is related to yeast-like symbionts from the planthoppers: Laodelphax striatellus, Nilaparvata lugens, and Sogetella furcifera. The second yeast was a member of the Candida genus, a group that has been identified in beetles and recently described in planthoppers. Microscopy revealed the presence of yeast in the fat body of P. saccharicida. The Candida yeast was cultured, and transformation was accomplished by electroporation of Candida albicans codon optimized plasmids, designed to integrate into the genome via homologous recombination. Transgenic lines conferred resistance to the antibiotic nourseothricin and expression of green fluorescent protein was observed in a proportion of the yeast cells. Stably transformed yeast lines could not be isolated as the integrative plasmids presumably replicated within the yeast without integration into the genome. If stable transformation can be achieved, then this yeast may be useful as an agent for a paratransgenic control of FLG.

Candida guilliermondii is an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. This yeast also remains an interesting biotechnological model for the industrial production of value-added metabolites. The recent whole-genome sequencing of the C. guilliermondii ATCC 6260 reference strain provides an interesting resource for elucidating new molecular events supporting pathogenicity, antifungal resistance and for exploring the potential of yeast metabolic engineering. In the present study, we designed an efficient transformation system for C. guilliermondii wild-type strains using both nourseothricin- and hygromycin B-resistant markers. To demonstrate the potential of these drug-resistant cassettes, we carried out the disruption and the complementation of the C. guilliermondii FCY1 gene (which encodes cytosine deaminase) known to be associated with flucytosine sensitivity in yeast. These two new dominant selectable markers represent powerful tools to study the function of a large pallet of genes in this yeast of clinical and biotechnological interest.

SUMMARY A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5–6.0 transformants per 108 cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture.

To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/ FRT recombination system from the yeast Saccharomyces cerevisiae for marker recycling. We tested this system in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation ( FRT - nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp ( Pcflp ) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT - nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional recombination system be generated in P. chrysogenum . As a proof of application of this system, we constructed a ΔPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/ FRT system, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/ FRT recombination system in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora . Here, we discuss the application of the optimized FLP/ FRT recombination system as a molecular tool for the genetic manipulation of filamentous fungi.

Yarrowia lipolytica is a non-conventional yeast of interest to the biotechnology industry. However, the physiology, metabolism, and genetic regulation of Y. lipolytica diverge significantly from more well-studied and characterized yeasts such as Saccharomyces cerevisiae. To develop additional genetic tools for this industrially relevant host, the piggyBac transposon system to enable efficient generation of genome-wide insertional mutagenesis libraries and introduction of scarless, footprint-free genomic modifications in Y. lipolytica. Specifically, we demonstrate piggyBac transposition in Y. lipolytica, and then use the approach to screen transposon insertion libraries for rapid isolation of mutations that confer altered canavanine resistance, pigment formation, and neutral lipid accumulation. We also develop a variety of piggyBac compatible selection markers for footprint-free genome engineering, including a novel dominant marker cassette (Escherichia coli guaB) for effective Y. lipolytica selection using mycophenolic acid. We utilize these marker cassettes to construct a piggyBac vector set that allows for auxotrophic selection (uracil or tryptophan biosynthesis) or dominant selection (hygromycin, nourseothricin, chlorimuron ethyl, or mycophenolic acid resistance) and subsequent marker excision. These new genetic tools and techniques will help to facilitate and accelerate the engineering of Y. lipolytica strains for efficient and sustainable production of a wide variety of small molecules and proteins.

Gene deletion in Schizophyllum commune is hampered by a low incidence of homologous integration. As a consequence, extensive screening is required to identify a transformant with the desired genotype. To alleviate this and to facilitate the assembly of deletion plasmids, vector pDelcas was constructed. This construct has a set of restriction sites, which allows for directional cloning of the flanking sequences at both sides of a nourseothricin resistance cassette. Moreover, it contains a phleomycin resistance cassette elsewhere in the plasmid, which is used to screen for transformants with an ectopic integration of the pDelcas derivative. The use of pDelcas derivatives in combination with an improved PCR screening protocol permitted the efficient identification of S. commune deletion strains. This procedure may also function in other basidiomycetes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11274-010-0356-0) contains supplementary material, which is available to authorized users.

Phleomycin is mutagenic by introducing double-strand breaks in DNA. The ble gene of Streptoalloteychus hindustanus , which confers resistance to this substance, is widely used as a selection marker for transformation. Schizophyllum commune grows on 25 μg of phleomycin ml −1 after introduction of a resistance cassette based on the ble gene. However, we here report that growth of resistant colonies on this concentration of phleomycin resulted in aberrant colony morphologies. Apparently, phleomycin was mutagenic despite acquired resistance. Therefore, a new selection system was developed based on resistance to the antibiotic nourseothricin. However, the transformation efficiency was tenfold lower than that obtained with phleomycin as a selection agent. This low transformation efficiency could be rescued by addition of a nonselective concentration of phleomycin during protoplast regeneration. This was accompanied by a higher incidence of single-copy integrations and with an increase of expression of key genes involved in double-strand break repair. Taken together, we conclude that the effect of a nonselective concentration of phleomycin strongly resembles the effect of restriction enzyme-mediated integration (REMI) but, unlike REMI, it does not depend on the presence of a target restriction site.

Candida parapsilosis is a major cause of human disease, yet little is known about the pathogen’s virulence. We have developed an efficient gene deletion system for C. parapsilosis based on the repeated use of the dominant nourseothricin resistance marker (caSAT1) and its subsequent deletion by FLP-mediated, site-specific recombination. Using this technique, we deleted the lipase locus in the C. parapsilosis genome consisting of adjacent genes CpLIP1 and CpLIP2. Additionally we reconstructed the CpLIP2 gene, which restored lipase activity. Lipolytic activity was absent in the null mutants, whereas the WT, heterozygous, and reconstructed mutants showed similar lipase production. Biofilm formation was inhibited with lipase-negative mutants and their growth was significantly reduced in lipid-rich media. The knockout mutants were more efficiently ingested and killed by J774.16 and RAW 264.7 macrophage-like cells. Additionally, the lipase-negative mutants were significantly less virulent in infection models that involve inoculation of reconstituted human oral epithelium or murine intraperitoneal challenge. These studies represent what we believe to be the first targeted disruption of a gene in C. parapsilosis and show that C. parapsilosis–secreted lipase is involved in disease pathogenesis. This efficient system for targeted gene deletion holds great promise for rapidly enhancing our knowledge of the biology and virulence of this increasingly common invasive fungal pathogen.

The genus Eremothecium contains dimorphic and filamentous fungal species, most notably Eremothecium sinecaudum (Holleya sinecauda), a dimorphic plant pathogen, which was isolated from mustard seeds, and Eremothecium gossypii (Ashbya gossypii), a filamentous fungus, which is well known for its ability to produce riboflavin. In this study, we present the initial molecular characterization of another Eremothecium species classified as Eremothecium coryli. E.coryli is a dimorphic fungus. We have developed, based on previously described reagents, a transformation system for E. coryli using kanMX and NATMX3 as dominant selectable marker genes on freely replicating plasmids conferring resistance to the antibiotics G418 and nourseothricin, respectively. As reporter genes we could introduce lacZ and GFP, which were controlled either by the AgTEF1 promoter or by regulatable MET promoters derived from the A. gossypii and Saccharomyces cerevisiae MET3 genes. These newly established tools will allow a detailed comparison of different growth modes in filamentous or dimorphic species within the genus Eremothecium.

The results present evidence for the important role of phosphate-mediated regulation of the nourseothricin biosynthesis by the processes of phosphate limitation and release of phosphate. Also, higher initial concentrations of phosphate were found to have a strong inhibitory effect on the biosynthesis of nourseothricin. It is concluded that the initial phosphate concentration was the primary target for the biometrical optimization of the fermentation medium. The presence of zinc ions neutralized the negative effect of high initial concentrations of phosphate which was also strongly influenced by the regime of sterilization.

Mutants with various levels of resistance to streptothricin were isolated from Escherichia coli K12, strain A19 after mutagenesis with N-methyl-N'-nitroso-guanidine and ethylmethane-sulfonate. Nourseothricin, a mixture of streptothricin F. and D was the selection agent. Spontaneous resistant mutants could not be found. The streptothricin-resistant mutant E. coli A19 Stcr 2/2/1 shows cross-resistance to some of the aminoglycoside antibiotics investigated, but no cross-resistance to chloramphenicol and chlortetracyclin. These results indicate similar mechanisms of action of streptothricin and aminoglycoside antibiotics.

We describe new heterologous modules for PCR-based gene targeting in the fission yeast Schizosaccharomyces pombe. Two bacterial genes, hph and nat, which display dominant drug-resistance phenotypes, are used as new selectable markers in these modules. Both genes have been used successfully in the budding yeast Saccharomyces cerevisiae, in which hph confers resistance to hygromycin B, while nat confers nourseothricin resistance (Goldstein and McCusker, 1999). Vector modules for gene disruption and C-terminal tagging with 3HA, 13Myc and GFP(S65T) are constructed using previously constructed pFA6a-MX6-derived plasmids (Bähler et al., 1998; Wach et al., 1997). In combination with the existing systems that are based upon the G418-resistance gene (kan), triple gene deletions or tags could be constructed. In addition a vector for one-step integration of a monomeric RFP (mRFP) to the C-terminus of proteins of interest is developed. Finally, oligonucleotides that allow a simple marker switch from kan to hph or nat, and vice versa, are described. The new constructs developed here should facilitate post-genomic molecular analysis of protein functions in fission yeast.

The ease of construction of multiple mutant strains in Schizosaccharomyces pombe is limited by the number of available genetic markers. We describe here three new cassettes for PCR-mediated gene disruption that can be used in combination with commonly used fission yeast markers to make multiple gene deletions. The natMX6, hphMX6 and bleMX6 markers give rise to resistance towards the antibiotics nourseothricin (NAT), hygromycin B and phleomycin, respectively. The cassettes are composed of exogenous sequences to increase the frequency of integration at targeted loci, and have a structure similar to the commonly used pFA6a-kanMX6 modular plasmid system. This allows a simple exchange of the kanMX6 marker in existing strains with any of the three new cassettes. Alternatively, oligonucleotide primers designed for the modular kanMX6 cassettes can be used to make the transforming PCR fragments for gene disruption. We illustrate the construction of a mutant strain with six independent gene disruptions, using the novel antibiotic cassettes in combination with existing genetic markers., Evaluation Studies, Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin ( NAT )-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37°C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37°C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1 , whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium -mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu + transport into the secretory pathway is compromised, depriving laccase and other Cu + -dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium -mediated transformation is more efficient and generates exclusively stable insertion mutations.

Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S. carlsbergensis (syn. of S. pastorianus) brewing yeast as well as in Saccharomyces kluyveri. As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option. Furthermore, the lacZ system for analyzing gene expression was included in the system.

Plasmid pRE25 from Enterococcus faecalis transfers resistances against kanamycin, neomycin, streptomycin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, tylosin, chloramphenicol, and nourseothricin sulfate by conjugation in vitro to E. faecalis JH2-2, Lactococcus lactis Bu2, and Listeria innocua L19. Its nucleotide sequence of 50237 base pairs represents the largest, fully sequenced conjugative multiresistance plasmid of enterococci (Plasmid 46 (2001) 170). The gene for chloramphenicol resistance (cat) was identified as an acetyltransferase identical to the one of plasmid pIP501 of Streptococcus agalactiae. Erythromycin resistance is due to a 23S ribosomal RNA methyl transferase, again as found in pIP501 (ermB). The aminoglycoside resistance genes are packed in tandem as in transposon Tn5405 of Staphylococcus aureus: an aminoglycoside 6-adenyltransferase, a streptothricin acetyl transferase, and an aminoglycoside phosphotransferase.). Identical resistance genes are known from pathogens like Streptococcus pyogenes, S. agalactiae, S. aureus, Campylobacter coli, Clostridium perfringens, and Clostridium difficile. pRE25 is composed of a 30.5-kbp segment almost identical to pIP501. Of the 15 genes involved in conjugative transfer, 10 codes for putative transmembrane proteins (e.g. trsB, traC, trsF, trsJ, and trsL). The enterococcal part is joined into the pIP501 part by insertion elements IS1216V of E. faecium Tn1545 (three copies), and homologs of IS1062 (E. faecalis) and IS1485 (E. faecium). pRE25 demonstrates that enterococci from fermented food do participate in the molecular communication between Gram-positive and Gram-negative bacteria of the human and animal microflora.

Genetic manipulation of fungi requires quick, low-cost, efficient, high-throughput and molecular tools. In this paper, we report 22 entry constructs as new molecular tools based on the Gateway technology facilitating rapid construction of binary vectors that can be used for functional analysis of genes in fungi. The entry vectors for single, double or triple gene-deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, entry vectors containing green fluorescent (GFP) or red fluorescent (RFP) in combination with hygromycin, geneticin or nourseothricin selection markers were generated. The latter vectors provide the possibility of gene deletion and simultaneous labelling of the fungal transformants with GFP or RFP reporter genes. The applicability of a number of entry vectors was validated in Zymoseptoria tritici, an important fungal wheat pathogen.

A liquid chromatographic method has been developed for the quantification of nourseothricin (NTC) in fermentation broths. The two major components D and F of the nourseothricin complex, were separated by HPLC using a C8 bond silica gel column and an aqueous ion pair solvent consisting of 0.02% octane-1-sulfonic acid sodium salt, 0.05% trifluoroacetic acid and 43% acetonitrile. The individual components, as well as unknown minor constituents, were detected by UV 210 nm. It was decided to quantify NTC based on the sum of the peak areas of D and F. The analytical response was linear over a concentration range of 10–80 μg/mL.

Cryptococcus neoformans is an excellent model system for studies on the molecular pathogenesis of fungal infections. There is only one dominant selectable market that can be used in the transformation of this organism, and we wanted to develop another. We found that various strains of C. neoformans are very sensitive to the aminoglycoside antibiotic nourseothricin, and that spontaneous resistance to this drug must be an extremely rare event. Resistance to nourseothricin is conferred by the product of the nourseothricin acetyltransferase gene (nat1) from Streptomyces noursei. In order to express this gene in C. neoformans, we created a fusion construct by driving expression of natl with the promoter sequence from a C. neoformans actin gene. Biolistic transformation of the serotype A C. neoformans strain H99 and the serotype D strain JEC21 with this construct resulted in transformation efficiencies of approximately 1,000 transformants microg(-1) of DNA and 20 transformants microg(-1) of DNA, respectively. Southern blots were performed using DNA from some of the H99 transformants, and this confirmed that all of the resistant isolates had the construct integrated in a random fashion within the genome. There was no cross-resistance of the nourseothricin-resistant transformants to hygromycin B, which is the other antibiotic used as a dominant selection marker in C. neoformans. The development of nourseothricin resistance as a second dominant selectable market will be helpful in future molecular studies on this important pathogenic fungus.

Protozoan parasites of the genus Leishmania are the etiologic agents of a spectrum of human diseases referred to as leishmaniasis. Leishmania alternates life stage between an intracellular amastigote form residing within the phagolysosome of vertebrate macrophage and an extracellular promastigote form living in the gut of sand flies. Molecular characterization of the genome of Leishmania has been greatly facilitated by the development of stable transfection methods, expression vectors and gene knockouts [1,2]. Stable transfection of Leishmania and trypanosomes requires the use of dominant selectable markers, and a number of independent markers have been used successfully. These include neomycin phosphotransferase (NEO) [3]; hygromycin phosphotransferase (HYG) [4], streptothricin acetyltransferase (SAT) [5], puromycin acetyltransferase (PAC) [6] and the bleomycin binding protein from Streptoalloteichus hindustanus (PHLEO) [6], which confer resistance to G418, hygromycin B, nourseothricin, puromycin and phleomycin respectively. In addition, overexpression of N-acetylglucosamine-1-phosphate transferase (NAGT) or dihydrofolate reductasethymidylate synthase (DHFR-TS) can confer resistance to tunicamycin and methotrexate respectively, allowing their use as selectable markers on episomal vectors [7,8]. Since Leishmania is diploid and lacks an experimentally manipulable sexual cycle, generation of null mutants requires the inactivation of two alleles [4]. Typically this is accomplished by two rounds of targeting with independent selectable markers, with a third marker needed to generate an ‘add-back’ construct restoring gene expression; this is an essential control for biological function [9]. While the use of loss of heterozygosity approaches can reduce the number of markers needed for null mutant construction [10], with the current repertoire of markers one can manipulate Abbre6iations: BSD, gene encoding blasticidin deaminase. * Corresponding author. Tel.: +1-314-7472630; fax: +1314-7472634. E-mail address: beverley@borcim.wustl.edu (S.M. Beverley)

Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.

Molecular analysis of parasite genomes will require new molecular genetic tools. The nat1 gene of Streptomyces noursei encodes nourseothricin acetyltransferase, conferring resistance to the aminoglycoside antibiotic nourseothricin. Electroporation of nat1 cassettes into RH or Prugniaud strains of Toxoplasma gondii allows for selection of stable nourseothricin-resistant clones.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system., Swiss National Science Foundation (www.snf.ch). École polytechnique fédérale de Lausanne (www.epfl.ch). Cancer Research UK [CRUK] C147/A6058.

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata.

In the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic ‘scars’. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant natMX6 (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved S. pombe cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions.

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.

Development of Nourseothricin N-acetyl transferase (NAT) as a selection marker for mammalian cells is described. Mammalian cells are acutely susceptible to Nourseothricin, similar to the widely used drug Puromycin, and NAT allows for quick and robust selection of transfected/transduced cells in the presence of Nourseothricin. NAT is compatible with other selection markers puromycin, hygromycin, neomycin, blasticidin, and is a valuable addition to the repertoire of mammalian selection markers.

The pLEX series of vectors was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania . These pUC-based constructs contain one of three independent selectable markers and a multiple cloning site inserted between the upstream and downstream untranslated regions of the previously cloned Leishmania major HEXBP gene. Selection was based on resistance to the aminoglycosides, hygromycin B and neomycin, and to nourse-othricin, a novel independent selectable marker for transfection of Leishmania . The vectors were introduced into Leishmania promastigotes by electroporation and were maintained as extrachromosomal circular concatemers containing between four and eight repeat units of the pLEX monomer. To demonstrate the efficient expression of cloned exogenous genes using the pLEX system, promastigotes were transfected with a pLEX construct that contained a second drug-resistant selectable marker gene cloned into the expression site, and clones were obtained that grew on media containing two antibiotics. These vectors, together with the novel selectable marker, will further facilitate the molecular analysis of gene expression in Leishmania .

The red yeasts of the Pucciniomycotina have rarely been transformed with DNA molecules. Transformation methods were recently developed for a species of Sporobolomyces, based on selection using uracil auxotrophs and plasmids carrying the wild-type copies of the URA3 and URA5 genes. However, these plasmids were ineffective in the transformation of closely related species. Using the genome-sequenced strain of Rhodotorula graminis as a starting point, the URA3 and URA5 genes were cloned and tested for the transformation ability into different Pucciniomycotina species by biolistic and Agrobacterium-mediated transformations. Transformation success depended on the red yeast species and the origin of the URA3 or URA5 genes, which may be related to the high G + C DNA content found in several species. A new vector was generated to confer resistance to nourseothricin, using a native promoter from R. graminis and the naturally high G + C nourseothricin acetyltransferease gene. This provides a second selectable marker in these species. Targeted gene disruption was tested in Sporobolomyces sp. IAM 13481 using different lengths of homologous DNA with biolistic and Agrobacterium transformation methods. Both DNA delivery methods were effective for targeted replacement of a gene required for carotenoid pigment biosynthesis. The constructs also triggered transgene silencing. These developments open the way to identify and manipulate gene functions in a large group of basidiomycete fungi.

Targeted gene inactivation is an important method to investigate gene function. In the diploid yeast Candida albicans, the generation of homozygous knock-out mutants requires the sequential replacement of both alleles of a gene by a selection marker. Targeted gene deletion is often performed in auxotrophic host strains, which are rendered prototrophic after the insertion of appropriate nutritional marker genes into the target locus. The SAT1-flipping strategy described in this chapter allows gene deletion in prototrophic C. albicans wild-type strains with the help of a recyclable dominant selection marker. The SAT1 flipper cassette used for this purpose consists of the caSAT1 marker, which confers resistance to the antibiotic nourseothricin, and the caFLP gene, which encodes the site-specific recombinase FLP. The addition of flanking sequences of the target gene allows specific genomic insertion of the SAT1 flipper cassette by homologous recombination and selection of nourseothricin-resistant transformants. Expression of the FLP recombinase results in subsequent excision of the cassette, which is bordered by direct repeats of the FLP recognition sequence FRT, from the genome. The homozygous mutants obtained after two rounds of insertion and recycling of the SAT1 flipper cassette differ from the wild-type parental strain only by the absence of the target gene and can be used for the inactivation of additional genes and the generation of complemented strains using the same strategy.

Although Ustilago maydis is readily amenable to molecular genetic experimentation, few antibiotic-resistance markers are available for DNA-mediated transformation. This poses constraints on experiments involving targeted gene disruption and complementation. To address this problem, we constructed vectors using one of three additional genes as dominant selectable markers for transformation. Two genes, sat-1 (encoding streptothricin acetyltransferase) and Sh-ble (encoding a phleomycin-resistance polypeptide), are of bacterial origin and have been engineered for expression in Ustilago sp. The third gene encodes an allele of U. maydis beta-tubulin that confers resistance to the fungicide benomyl.

Electrostimulation has been an established method in medicine for many years. However, the application of electrostimulation to fermentation processes in biotechnology is a recent development. Although there are many publications 111 and patents in this field, only a few applications on the industrial scale have been reported. Electrostimulation in biology has been reviewed by Berg [1,21. Examples of electrostimulation in biotechnology are the increase in CO, formation at Succharomyces cerevisiae [31 and the influence of pulsatile electromagnetically induced currents (PEMIC) on the production of the antibiotic Nourseothricin from Streptomyces noursei [4]. One reason why electrostimulation is seldom applied in fermentation processes may be the complicated mechanisms and the unknown limiting step on the molecular level of most microbial fermentation processes, particularly in the case of syntheses of extracellular products. We are interested in the production of fungal cellulase which is a very complex system. The limiting step in the formation and secretion of the cellulase enzyme complex is governed by different biochemical and genetic control mechanisms, mainly the induction of enzyme formation and carbon catabolite repression. Carbon catabolite repression suppresses cellulase formation even if the concentration of easily utilizable sugars in the fermentation medium is very low, e.g. less than 20 mg 1-r of glucose. The mechanism of these regulatory processes on the molecular level is not yet completely understood. Many fungal mutant strains with improved cellulase formation have altered regulation of cellulase formation [5,6]. By using mutants with “switched off’ carbon catabolite repression it is possible to enhance cellulase formation significantly if the induction is optimal. We have previously developed such a Tricho

We have determined the nucleotide (nt) sequence of nat1, a gene encoding nourseothricin (Nc) acetyltransferase (AT) from Streptomyces noursei, and its transcriptional start point (tsp). The nt sequence upstream from the coding region is completely different from that of the stat gene (encoding streptothricin AT) from Streptomyces lavendulae [S. Horinouchi,K. Furuya, M. Nishiyama, H. Suzuki and T. Beppu, J. Bacteriol. 169 (1987) 1929–1937], even though the nt sequencesof the two genes and the deduced amino acid (aa) sequences of the two enzymes show a high degree of similarity. Another stat gene, derived from a Gram-negative plasmid, showed only deduced aa similarity, but not nt sequence similarity, to the above two. A database search for related aa sequences did not reveal any clear-cut homologies to other types of protein. A multiple aa sequence alignment of several ATs is presented.

Streptomyces lividans TK24 strains transformed with different recombinat derivatives of the vector plasmid pIJ487 were continuously cultivated in a chemostat. The first plasmid derivative contained the determinant for human interferon-alpha-1 (IFN) and the expression-secretion-unit (ESU) for this protein. In the second one a nourseothricin resistance determinant (ntc) was inserted additionally. The chemostat operated mainly at glucose limitation and low dilution rates. The structural and functional stabilities of the plasmids were shown to depend on the selection pressure. The host mutants enriched in the chemostat differed from the parental strain with respect to the growth pattern of aerial and submerged mycelium, the spore formation, and the formation and secretion of pigments and enzymes. Some highly stable host-vector systems could be selected. The plasmids' genotype influenced the growth pattern of the host mutants enriched in the chemostat in dependence on the limitation conditions as well as the stability of plasmid inheritance, plasmid structure and pigment formation in these mutants.

Seventy-two Enterococcus faecium isolates of different origins highly resistant to nourseothricin and streptomycin were studied. Sequencing of a genomic fragment from two isolates identified a gene cluster, aadE– sat4 – aphA-3 , which has been isolated recently in staphylococci and Campylobacter coli . Patterns of digested PCR products of aadE–sat4–aphA-3 were identical for all isolates.

Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2-containing plasmids or single-copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT-1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild-type C. albicans. We used PCR to fuse CaHygB or SAT-1 to approximately 1 kb of 5′ and 3′ noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild-type C. albicans strains. Homologous targeting frequencies were approximately 50–70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates. Copyright © 2010 John Wiley & Sons, Ltd.

Summary The polycationic antibiotic, nourseothricin, represents a mixture of several streptothricins, mainly D and F. The molecular weight of the latter compound amounts to 486. Obviously, although very slowly, it can pass the outer membrane via the porin pores. It has been shown earlier that nourseothricin is able to generate some kind of channels into the outer membrane through which it can pass the cell wall. On the other hand, there were indications that resistant strains containing a streptothricin-inactivating acetyl transferase possess an additional protecting system, namely a reduced penetrability of the outer membrane. In this study, it could be shown that such strains indeed could be rendered sensitive by damaging the barrier function of the outer membrane.

Dermatophytes are filamentous fungi that colonize the keratinized layer of human and animal skin. The availability of several selectable markers for dermatophyte gene manipulation would provide important tools to understand the genetic properties of this fungal group. In this study, we report the nourseothricin resistance gene nat1 that confers resistance to the aminoglycoside antibiotic nourseothricin as a dominant marker in Trichophyton mentagrophytes. The NAT cassette was introduced into T. mentagrophytes by the Agrobacterium tumefaciens-mediated transformation (ATMA) method. Transformation occurred at a frequency of 78 transformants per 1 x 10(7) cells. Molecular analysis showed integration of the NAT cassette into the genomic DNA of T. mentagrophytes. This study presents the nourseothricin resistance gene nat1 as a useful selectable marker for selection of T. mentagrophytes.

Ustilago maydis is an important model fungal organism for diverse studies. Little improvement has been made in the method for its transformation since the PEG-mediated transfection of spheroplasts that was reported more than 20years ago. We have constructed binary T-DNA vectors carrying Hygromycin and Nourseothricin resistance gene cassettes and have developed a highly efficient method for transformation of this fungus based on Agrobacterium tumefaciens-mediated transformation (ATMT). Through a series of optimization, at least 1x10(4) Hygromycin B resistant colony forming units (CFU) have been achieved on each 90mm agar plate using 10(6) sporidia. Optimal pH value for ATMT is approximately 5.6. Approximately 96% Hygromycin B-resistant transformants contain a single-copy T-DNA inserted into the nuclear genome. Analysis of 204 T-DNA flanking sequences showed that 15.2% of them were found in the coding sequences and a further 37.25% within 0.5kb from the coding sequences at the 5' UTR or promoter regions. In addition, a method for preparation and preservation of transformation-ready T-DNA donor and receptor cells has been developed allowing gene tagging experiments to be performed on-demand. An initial screening of 5000 mutants resulted in the identification of a putative farnesyl transferase beta subunit and a PRE6 homologue as new players of sexual mating in U. maydis.

Studies on the insectieidal activity of the antibiotic Nourseothricin The streptothricin antibiotic Nourseothricin has been tested against insects of different systematic groups. The observed insecticidal effect is manifested in a stop of development and later in the death of animals. This time depends on the dosage of the antibiotic and the temperature. Nourseothricin is an eating poison and it is resorpted by the midgut of insects. The mortality of animals is probably caused by the mortality of endosymbiotic bacteria and is connected with male nutrition and poisoning depending on toxic metabolic products. Zusammenfassung Das zur Streptothricingruppe gehorende Antibiotikum Nourseothricin zeigte gegenuber Insekten unterschiedlicher systematischer Zugehorigkeit eine abtotende Wirkung, die sich zunachst im Entwicklungsstillstand und spater im Absterben der Tiere auserte. Die Zeitdauer bis zum Tode der Tiere ist sowohl dosis- als auch temperaturabhangig. Nourseothricin wirkt als Frasgift und wird vom Insektendarm resorbiert. Der Wirkungsmechanismus beruht wahrscheinlich auf der Abtotung von endosymbiontischen Bakterien und allgemeintoxischen Effekten und ist mit einer Mangelernahrung und Vergiftung durch angereicherte toxische Stoffwechselprodukte verbunden.