36000, Guanajuato, Gto., MÃxico. Received: 13 April 2005 / Accepted: 17 October 2005. Abstract. Mucor circinelloides transformants prototrophic to leucine and ...
CURRENT MICROBIOLOGY Vol. 52 (2006), pp. 178–181 DOI: 10.1007/s00284-005-0088-9
Current Microbiology An International Journal ª Springer Science+Business Media, Inc. 2006
Transformation of Mucor circinelloides with Autoreplicative Vectors Containing Homologous and Heterologous ARS Elements and the Dominant Cbxr Carboxine-Resistance Gene R. Ortiz-Alvarado, G. A. Gonzalez-Hernandez, J. C. Torres-Guzman, J. F. Gutierrez-Corona Instituto de Investigaci'n en Biolog)a Experimental, Facultad de Qu)mica, Universidad de Guanajuato, Noria Alta s/n, Apartado Postal 187, 36000, Guanajuato, Gto., M7xico Received: 13 April 2005 / Accepted: 17 October 2005
Abstract. Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu+ Cbxr) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbxr (carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 l plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65–120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.
Members of the genus Mucor have proved to be of basic interest for their use as models in the study of different processes such as the physiological and biochemical basis of morphogenesis [10, 12] or the regulation of carotenoid synthesis [11]. They are also of applied importance because of the production by certain species of a number of industrially important enzymes [6, 13], as well as for their use in steroid bioconversion [9]. The use of Mucor circinelloides in the production of g-linolenic acid, a fatty acid important for human nutrition, is also under investigation [17, 21]. These aspects and the difficulties in performing formal genetic analysis have increased interest in the development of molecular tools for the genetic manipulation of these organisms. Recent progress in this direction has been attained through the development of an expression vector for recombinant protein production in M. circinelloides [22]. Autoreplicative properties of vectors in M. circinelloides [1, 15] have been shown to be due to auton-
omously replicating sequences (ARS), which were first described in the ascomycete yeast Saccharomyces cerevisiae [18, 20]. Until now, all the systems for the transformation of M. circinelloides have made use of auxotrophic recipient strains, either leucine [19], methionine [1], or uracil [4] requiring mutants, which could be complemented by the corresponding cloned wild-type gene. Other useful genetic markers that could be exploited for molecular genetic studies in different Mucor species are dominant selectable markers, which confer fungicide or antibiotic resistance to sensitive strains in virtually any medium. Recently, a geneticinbased dominant selection system for M. circinelloides and other zygomycetes has been described [2]. In this work, we report the use of heterologous ARS elements and the cbxr (carboxine-resistance) gene of Ustilago maydis [8] as a selectable marker for the transformation of M. circinelloides. Materials and Methods
Correspondence to: J.F. Gutierrez-Corona; email: felixg@quijote. ugto.mx
Strains and media. The M. circinelloides strain R7B, a leucine auxotroph [14], was used as the recipient in all transformation experiments. M. circinelloides was grown at 28C in minimal medium,
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R. Ortiz-Alvarado et al.: Transformation of M. circinelloides to Carboxine Resistance Table 1. Plasmid constructs employed Plasmid
Harbored gene(s) and element(s)
Reference
pLeu4 pGG37
M. circinelloides leuA and ARS M. circinelloides leuA and ARS; U. maydis cbxr and ARS U. maydis cbxr, 2 lm ARS from S. cerevisiae U. maydis cbxr, 2 lm ARS from S. cerevisiae M. circinelloides leuA and ARS U. maydis cbxr and ARS
[15] This work
pGG39 pGG43 pCbx122
This work This work [8]
Table 2. Transformation of M. circinelloides to leucine independence or carboxine resistance with plasmids bearing homologous or heterologous ARS elements Transformation frequencya when selecting for: Vector Untransformed control pLeu4 pGG43 pGG37 pGG39
Leu + phenotypeb
Cbxr phenotypeb
Relative transformation efficiencyc
0 332 € 20.8 545 € 26.4 697 € 29.8 —
0 0 532 € 31.0 705 € 35.0 170 € 16.5
— 1.0 2.1 1.6
a Transformation frequency is expressed as the number of Leu+ or Cbxr transformants per microgram of plasmid DNA per 107 cells. Values are the mean and standard deviations of three independent experiments. b Leu+ transformants were selected in minimal medium; Cbxr transformants were selected in minimal medium supplemented with leucine and carboxine. c Relative transformation efficiency was calculated from the data of Leu+ transformants frequency.
alone or supplemented with 50 lg/mL leucine, or in complex YPG medium [3], as required. Carboxine resistance was tested in minimal medium supplemented with 424 lM carboxine. Escherichia coli DH5a was used for bacterial transformation and propagation of plasmids as described [16].
ARS from S. cerevisiae; in addition, plasmid pGG43 contains the M. circinelloides leuA gene and ARS element. Plasmid pGG39 was constructed inserting the Eco RI-BamHI 2.7-kbp fragment from plasmid pCBX122; plasmid pGG43 was derived from pGG39 inserting the PstI 4.5-kbp fragment from plasmid pLeu4.
DNA isolation. Plasmid DNA was obtained from E. coli by the alkaline lysis method [17]. Total DNA was isolated from M. circinelloides mycelium using the method previously described for RNA isolation from mammalian cells [16], with the modifications as indicated [7].
Hybridization procedures. Labeling, hybridization, and detection were carried out using a nonradioactive commercial kit (Boehringer, Mannheim, FRG), according to standard protocols [16].
Transformation of bacteria. E. coli cells were made competent by the CaCl2 method and transformed to ampicillin resistance following standard procedures [16]. Transformation of M. circinelloides. Conditions for protoplast production and transformation were as described previously [22]. Plasmids. Plasmid constructs used are shown in Table 1. Plasmid pLeu4 is an autonomously replicating vector containing the M. circinelloides leuA gene and an adjacent ARS element [15]. Plasmid pGGG37 is a pUC19-derived vector, which contains the M. circinelloides leuA gene and adjacent ARS element, as well as U. maydis cbxr gene and ARS element. The construct was made using the Eco RI-BamHI 2.7 kbp and SspI 0.3-kbp fragments from plasmid pCBX122, bearing the U. maydis cbxr gene and ARS element, respectively [8] and the PstI 4.5-kbp fragment from plasmid pLeu4, bearing the M. circinelloides leuA gene and ARS element. Plasmids pGG39 and pGG43 were derived from the S. cerevisiae plasmid YEp52 [5] and both contain the U. maydis cbxr gene and the 2 lm
Results and Discussion Transformation of M. circinelloides to leucine independence and carboxine resistance with plasmids bearing different ARS elements. Protoplasts of the M. circinelloides Leu- strain R7B transformed with plasmids pLeu4, pGG37, and pGG43 gave rise to leucine-independent colonies; control experiments using protoplasts without DNA gave no transformants (Table 2). Most (>90%) of the Leu+ transformants bearing plasmids PGG37 or pGG43 exhibited capacity to grow in the presence of carboxine (data not shown), whereas the transformants obtained with control plasmid pLeu4 or the untransformed strain R7B were unable to grow in its presence (Table 2). Figure 1 shows the Leu+ Cbxr phenotype of transformants obtained with plasmids
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Fig. 1. Cbxr phenotype of M. circinelloides transformants bearing plasmids pGG43 or pGG37. Transformants bearing plasmids pLeu4 (1), pGG43 (2), and pGG37 (3) were incubated for 5 days in minimal medium without (A) or with (B) 424 lM carboxine.
pGG37 and pGG43 and the Leu+ Cbxs phenotype of Leu+ transformants obtained with plasmid pLeu4. These results indicated that the Cbxr gene from U. maydis is expressed in M. circinelloides conferring resistance to the fungicide, which suggested that this gene could be used as selectable marker for the transformation of the fungus. This suggestion was probed by transformation experiments performed under conditions selective for carboxine resistance but nonselective for leucine prototrophy. As shown in Table 2, carboxine-resistant transformants could be directly selected in the presence of the fungicide in minimal medium supplemented with leucine. Plasmids pGG37, pGG43, and pGG39 produced 705 € 50, 530 € 35, and 170 € 16.5 carboxine-resistant transformants per microgram DNA, respectively. The data of Table 2 indicated that the relative frequency of transformation with plasmids pGG37 and pGG43, which contain the M. circinelloides ARS element as well as an ARS sequence from U. maydis (plasmid pGG37) or from the 2-lm plasmid of S. cerevisiae (plasmid pGG43) was increased in about 120% and 65%, respectively, as compared to plasmid pLeu4, which contains the M. circinelloides ARS element alone. These findings indicated that the presence of more than one ARS element in plasmid molecules led to an improvement in their transforming capabilities, perhaps positively influencing the replication of the plasmids. It has been shown that heterologous ARS from different sources, including Xenopus, Tetrahymena, and human cells can actively promote autonomous replication of plasmids in S. cerevisiae [21]. This might suggest that the improvement of the transforming capabilities of plasmid vectors from different eukaryotic cells could be achieved using homologous and/or heterologous ARS elements.
Characterization of transformants. Mitotic stability studies indicated that in transformants harboring plasmids pGG37 or pGG43 leucine prototrophy and carboxine-resistance traits were unstable, because they were nearly lost after two vegetative cycles in nonselective medium (data not shown). This instability and the co-segregation of the Leu+ and Cbxr phenotypic traits indicated the presence of the leuA and cbxr genes in an autonomous replicating element. The presence of plasmid molecules containing the U. maydis cbxr gene within transformant clones was confirmed by Southern hybridization analysis (Fig. 2). Transformants selected as Leu+ with plasmid pGG37 (lane 4) or pGG43 (lane 5) gave a strong hybridization signal when probed with the PstI 1.9 kb fragment of plasmid pCbx122, bearing the cbxr gene and used as a positive control (lane 2). In these conditions, DNA from the untransformed strain R7-B gave a weak hybridization signal (lane 3), probably due to a poor hybridization of the probe to the corresponding wild-type gene in M. circinelloides genome. A similar hybridization pattern was obtained with DNA from Cbxr transformants obtained with plasmids pGG37 or pGG43; in addition, the DNA of these transformants as well as that from transformants selected as Cbxr with plasmid pGG39 revealed the presence of the U. maydis cbxr gene when tested by polymerase chain reaction (data not shown). Further evidence for the presence of an autonomous replicating plasmid in the transformants bearing vectors pGG37 or pGG43 was obtained by the rescue of these plasmids from ampicillin-resistant bacterial colonies, produced after transformation of E. coli with undigested total DNA from the M. circinelloides transformants (data not shown).
R. Ortiz-Alvarado et al.: Transformation of M. circinelloides to Carboxine Resistance
5.
6.
7.
8.
9. 10.
11. Fig. 2. Southern blot analysis of total DNA prepared from M. circinelloides transformants. Lane 1, plasmid pGG39; lane 2, plasmid pCbx122; lane 3, untransformed strain R7B; lane 4, Leu+ selected transformant strain bearing pGG37 plasmid; lane 5, Leu+ selected transformant strain bearing pGG43 plasmid. The blots were probed with the 1.9-kb PstI fragment of pCbx122 plasmid, containing the U. maydis cbxr gene. DNA samples from M. circinelloides strains were digested with PstI.
Taken together, the results of this work indicated that the U. maydis Cbxr gene and the heterologous ARS elements tested are functional in M. circinelloides; thereby they could be useful for the development of new molecular tools for the genetic manipulation of Zygomycete fungi.
12. 13.
14.
15.
16.
17. ACKNOWLEDGMENTS This work was supported by Consejo Nacional de Ciencia y Tecnolog)a (CONACyT) and Universidad de Guanajuato, Mexico. R.O.A. received a fellowship from CONACyT, Mexico.
Literature Cited 1. Anaya N, Roncero MIG (1991) Transformation of a methionine auxotrophic mutant of Mucor circinelloides by direct cloning of the corresponding wild type gene. Mol Gen Genet 230:449–455 2. Appel KF, Wolff AM, Arnau JA (2004) Multicopy vector system for genetic studies in Mucor circinelloides and other zygomycetes. Mol Genet Genomics 271:595–602 3. Bartnicki-Garc)a S, Nickerson WJ (1962) Nutrition, growth, and morphogenesis of Mucor rouxii. J Bacteriol 84:841–858 4. Benito EP, Diaz-Minguez JM, Iturriaga EA, Campuzano V, Eslava AP (1992) Cloning and sequence analysis of the Mucor circinelloides pyrG gene encoding orotidine-5¢-monophosphate decar-
18.
19.
20. 21.
22.
181
boxylase: use of pyrG for homologous transformation. Gene 116:59–67 Broach JR, Hicks JB (1980) Replication and recombination functions associated with the yeast plasmid, 2 lm circle. Cell 21:501–508 Godtfredsen SE (1990) Microbial lipases. In: Fogarty WM, Kelly CT (eds) Microbial enzymes and biotechnology. 2nd edn. London: Elsevier Applied Science Publishers, pp 255–274 GonzSlez-Hernandez GA, Herrera-Estrella L, Rocha-Ramirez V, Roncero MIG, Gutierrez-Corona JF (1997) Biolistic transformation of the Zygomycete Mucor circinelloides. Mycol Res 101:953– 956 Keon JPR, White GA, Hargreaves JA (1991) Isolation, characterization and sequence of a gene conferring resistance to the systemic fungicide carboxin from the maize smut pathogen, Ustilago maydis. Curr Genet 19:475–481 Madyastha KM, Srivatsan J (1987) Novel transformations of progesterone by a Mucor sp. Can J Microbiol 33:361–365 McIntyre M, Breum J, Arnau J, Nielsen J (2002) Growth physiology and dimorphism of Mucor circinelloides (syn. racemosus) during submerged batch cultivation. Appl Microbiol Biotechnol 58:495–502 Navarro E, Ruiz-Perez VL, Torres-Martinez S (2000) Overexpression of the crgA gene abolishes light requirement for carotenoid biosynthesis in Mucor circinelloides. Eur J Biochem 267:800–807 Orlowski M (1991) Mucor dimorphism. Microbiol Rev 55:234– 258 Outtrup H, Boyce COL (1990) Microbial proteinases and biotechnology. In: Fogarty WM, Kelly CT (eds) Microbial enzymes and biotechnology. 2nd edn. London: Elsevier Applied Science Publishers, pp 227–254 Roncero MIG (1984) Enrichment method for the isolation of auxotrophic mutants of Mucor using the polyene antibiotic Nglycosyl-polifungin. Carlsberg Res Commun 49:685–690 Roncero MIG, Jepsen LP, Strøman P, van Heeswijck R (1989) Characterization of a leuA gene and an ARS element from Mucor circinelloides. Gene 84:335–343 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press Serrano I, Lopes da Silva T, Carlos Roseiro J (2001) Ethanolinduced dimorphism and lipid composition changes in Mucor fragilis CCMI 142. Lett Appl Microbiol 33:89–93 Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039 Van Heeswijck R, Roncero MIG (1984) High frequency transformation of Mucor with recombinant plasmid DNA. Carlsberg Res Commun 49:691–702 Williamson HD (1985) The yeast ARS element, six years on: a progress report. Yeast 1:1–14 Wynn JP, Hamid AA, Li Y, Ratledge C (2001) Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina. Microbiol 147:2857–2864 Wolff AM, Arnau J (2002) Cloning of glyceraldehyde-3-phosphate dehydrogenase-encoding genes in Mucor circinelloides (Syn. racemosus) and use of the gpd1 promoter for recombinant protein production. Fung Genet Biol 35:21–29
