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A gene for carotene cleavage required for pheromone biosynthesis and carotene regulation in the fungus Phycomyces blakesleeanus
Fungal Genetics and Biology 49 (2012) 398–404
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A gene for carotene cleavage required for pheromone biosynthesis and carotene regulation in the fungus Phycomyces blakesleeanus Víctor G. Tagua a,1, Humberto R. Medina a,1, Raúl Martín-Domínguez b, Arturo P. Eslava b, Luis M. Corrochano a,⇑, Enrique Cerdá-Olmedo a,⇑, Alexander Idnurm c,⇑ a b c
Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41080 Sevilla, Spain Área de Genética, Departamento de Microbiología y Genética, Universidad de Salamanca, Edificio Departamental, 37007 Salamanca, Spain Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri–Kansas City, Kansas City, MO 64110, United States
a r t i c l e
i n f o
Article history: Received 9 December 2011 Accepted 8 March 2012 Available online 21 March 2012 Keywords: Apocarotenoid Beta-carotene Mucoromycotina Pheromone biosynthesis Zygomycete
a b s t r a c t Mating and sexual development in fungi are controlled by molecular mechanisms that are specific for each fungal group. Mating in Phycomyces blakesleeanus and other Mucorales requires pheromones derived from b-carotene. Phycomyces mutants in gene carS accumulate large amounts of b-carotene but do not enter the sexual process. We show that carS encodes a b-carotene-cleaving oxygenase that catalyzes the first step in the biosynthesis of a variety of apocarotenoids, including those that act as pheromones. Therefore carS mutants cannot stimulate their sexual partners, although they respond to them. CarS catalyzes the biosynthesis of a b-ring-containing apocarotenoid that inhibits the activity of the carotenogenic enzyme complex in vegetative cells and provides a feedback regulation for the b-carotene pathway. The carS gene product is a keystone in carotenogenesis and in sexual reproduction. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Phycomyces blakesleeanus is a saprophytic fungus used in many studies of physiology, environmental sensing, genetics and metabolism (Bergman et al., 1969; Cerdá-Olmedo, 2001; Cerdá-Olmedo and Lipson, 1987). Phycomyces and other Mucorales were the first organisms, outside Animals and Plants, known to reproduce sexually (Ainsworth, 1976; Blakeslee, 1904; Idnurm, 2011), but the study of their sexual processes is not as advanced as in the Ascomycete and Basidiomycete fungi (Lee et al., 2010) and other microorganisms. The heterothallic Mucorales are distinctive in the existence of two sexes that look identical and behave symmetrically (isogamy). The two sexes, called (+) and (), are determined by two allelic but dissimilar DNA segments carrying genes sexP and sexM, respectively, whose products are HMG-domain transcription factors (Gryganskyi et al., 2010; Idnurm et al., 2008; Lee et al., 2008; Li et al., 2011). Sexual interaction in Phycomyces and other Mucorales begins with morphological modification of hyphal tips into zygophores and involves an exchange of diffusible signals, the first pheromones discovered in any organism (Burgeff, 1924). These signals are apocarotenoids, derived from fragments of the yellow pigment ⇑ Corresponding authors. E-mail addresses: [email protected] (L.M. (E. Cerdá-Olmedo), [email protected] (A. Idnurm). 1 These authors contributed equally to this work.
Corrochano),
1087-1845/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2012.03.002
[email protected]
b-carotene (Austin et al., 1970; Caglioti et al., 1966; Schimek and Wöstemeyer, 2009), in contrast to the modified peptides used by Ascomycetes and Basidiomycetes (reviewed by Jones and Bennett, 2011). Phycomyces mycelia accumulate all-trans b-carotene synthesized by a carotenogenic enzyme complex (Aragón et al., 1976; Candau et al., 1991; De la Guardia et al., 1971; Sanz et al., 2002) composed of at least seven protein molecules that carry out three different catalytic activities and are the products of two genes, carB and carRA (Arrach et al., 2001; Ruiz-Hidalgo et al., 1997). The b-carotene content of Phycomyces is enhanced by blue light, sexual interaction and many chemicals, and is modified by mutations in various genes (Cerdá-Olmedo, 2001). Blue light is perceived by a photoreceptor and transcription factor complex (Idnurm et al., 2006; Sanz et al., 2009) and stimulates the biosynthesis of b-carotene through the transcription of the carB and carRA genes (Almeida and Cerdá-Olmedo, 2008; Ruiz-Hidalgo et al., 1997; Sanz et al., 2010). Apocarotenoids also increase the carotene content by increasing the transcription of the structural genes during mating (Almeida and Cerdá-Olmedo, 2008), in a separate response from zygophore formation (Kuzina and Cerdá-Olmedo, 2006). A common mechanism mediates the activation of the b-carotene pathway by mutational or chemical inhibition of the carotenogenic enzymes, by retinol or b-ionone added to the medium, and by mutation of carS (Bejarano and Cerdá-Olmedo, 1989; Bejarano et al., 1988; Eslava et al., 1974; Murillo and Cerdá-Olmedo, 1976).
V.G. Tagua et al. / Fungal Genetics and Biology 49 (2012) 398–404
Apocarotenoids of 18 and 15 carbon atoms have been isolated from the cultures of Phycomyces and other Mucorales. The recent identification of (2E,4E)-6-hydroxy-5-methylhexa-2,4-dienoic acid and other 7-carbon compounds as apocarotenoids (Barrero et al., 2011; Polaino et al., 2010) has shown that the 40-carbon bcarotene molecule is split into three fragments of 18, 7 and 15 carbon atoms, heads of the respective three families of apocarotenoids, the trisporoids, the methylhexanoids, and the cyclofarnesoids, that include those that act as pheromones. Outside of the Dikarya fungi, no gene specific to pheromone biosynthesis has been identified, that is, as mutated in a strain with impaired pheromone production. Here we identify the sequence of gene carS, we show that the carS gene product is a b-carotenecleaving oxygenase responsible for the biosynthesis of apocarotenoids in Phycomyces, and we clarify its role in the regulation of b-carotene production and pheromone biosynthesis. 2. Materials and methods 2.1. Strains and growth conditions Strain NRRL1555, the standard wild type, and other strains of Phycomyces blakesleeanus Bgff. (Mucoromycotina, Mucorales), described in Table S1, were grown at 22 °C in the dark on minimal agar plates inoculated with 104 heat-activated spores. Standard methods for this organism (Cerdá-Olmedo and Lipson, 1987) were followed, unless otherwise stated. Eschericha coli DH5-alpha was used for cloning plasmids. 2.2. Crosses and sexual development The zygospores from the crosses between strains A914 and UBC21, as established on V8 juice agar, germinated and produced germsporangia in about 2 months. A total of 45 progeny colonies, each from a separate germsporangium, was scored for auxotrophy (lysA), phototropism (madI), and restriction fragment polymorphisms after PCR (PCR–RFLP). Sequences for primers for PCR–RFLPs are provided in Table S2. SNPs that change target sites for restriction enzymes were identified after comparison of the genomic sequence of the () wild-type strain NRRL1555 and the Illumina genome sequence of the (+) wild-type strain UBC21. The position of SNPs along the draft of the P. blakesleeanus NRRL1555 genome sequence can be obtained from the JGI web page (http://genome. jgi-psf.org/Phybl1/Phybl1.home.html). The sexual development of the carS mutants was assayed after plating each of them and strain A56 at opposite ends of a potato agar plate. The plates were incubated 2 days at 22 °C and 5–7 days at 16 °C before inspection. 2.3. Sequencing carS alleles Phycomyces genomic DNA was used to amplify and sequence carS alleles in carS mutant strains. PCRs were performed with total genomic DNA, using primers listed in Table S3. PCR products were purified and sequenced by the chain-termination method. The sequence of carS is deposited in GenBank (accession GU339225). 2.4. Analysis of b-carotene and apocarotenoids Cultures were grown for 2 days and then exposed to light at 22 °C for 1 day or kept in the dark as a control. For light exposure the Petri dishes were incubated on a white surface under a set of four fluorescent lamps (Philips TLD 36 W/865, 4 W/m2 white light) placed 75 cm away. After light exposure mycelia were collected, frozen in liquid nitrogen, and lyophilized. Mycelia (about 100 mg
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each) were ground in a mortar with sea sand and a pestle, and extracted with petroleum ether (b.p. 40–60 °C). The extracts were centrifuged (8 min at 4000g), and their b-carotene content was calculated from their absorbance at 453 nm and the extinction coefficient (1 mg/L, 1 cm) = 259.2. The apocarotenoids were extracted and fractionated by reverse-phase HPLC as described (Polaino et al., 2012).
3. Results 3.1. Localization and identification of gene carS Phycomyces mutants in gene carS have a complex phenotype (Fig. 1): they overaccumulate b-carotene and react to strains of opposite sex with additional carotene production and changes in hyphal morphology, but fail to stimulate their partners and complete the sexual cycle (Kuzina et al., 2006; Salgado et al., 1989; Sutter, 1975). The structural genes for carotenogenesis carB and carRA are contiguous (Arrach et al., 2001) on contig 6 of the genome database (release version 1.1). They are genetically linked to carS at about 35 cM (Roncero and Cerdá-Olmedo, 1982) and to madI, a gene required for the phototropism of the fruiting body, at about 27 cM (Campuzano et al., 1995). Thus carS and madI may be linked, and this linkage could help identify carS. A cross was established between strains A914, genotype lysA madI (), and UBC21, a (+) wild type. The auxotrophy and the defective phototropism are easy to test. The two markers segregated independently (14 wild type, 10 lysA, 11 madI, and 10 lysA madI, in 45 progeny) confirming the lack of linkage (Eslava and Alvarez, 1996). When the same progeny were tested for the molecular markers available in contig 6 (Fig. 2), it was found that gene madI lies outside this contig, but not far from one of its ends. In order to identify additional DNA sequence linked to contig 6, the Phycomyces genome was analyzed using BLASTn with the 916-bp sequence at the end of contig 6. The rationale for this approach was based on the hypotheses that (i) contig assembly may be blocked by adjacent repeated DNA sequences and that regions that matched may be on an adjacent contig, and (ii) that repetitive elements can be chromosome-specific. One of the highest regions of similarity was in the middle of contig 32 and a molecular marker in contig 32 was linked to contig 6 (Fig. 2). Two putative genes in contig 32 caught our attention because of their possible relation to carotene metabolism. One of them (JGI ID# 85763) was similar to crgA of Mucor circinelloides, defined by carotene-overproducing mutants (Navarro et al., 2001), but its sequence was the same in a carS strain (M1) and in its parental strain (NRRL1554). The second sequence (ID# 79747) is one of five genes that we were studying independently because of their similarity to well-known carotene-cleaving oxygenases (Medina et al., 2011), and to gene tsp3, encoding a putative carotene oxygenase of Blakeslea trispora (Burmester et al., 2007). The sequence of gene with ID 79747 is carS, because each carS mutant tested (11 strains, six independent alleles) differed from the wild type in a single-base substitution in that sequence (Table 1, Fig. 3). In addition, the carS gene was sequenced in five carotene-overproducing strains (S275, S276, S101, S496, S498) derived from strain C115 by various crosses. All five strains contain the allele carS42 present in the original strain. The carS gene coding region is 2027 bp and contains two introns of 87 and 53 bp as determined by sequencing its cDNA. The predicted CarS protein is 628 amino acid residues in length (Fig. 3). The CarS protein sequence significantly coincides with those of proven b-carotene-cleaving oxygenases from animals, plants and fungi (Fig. 3). It includes four histidines and three glutamates or
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Fig. 1. Strains with mutations in carS are sterile and overproduce b-carotene. Left. Mycelia of the standard wild type (NRRL1555, mating type ) and an isogenic wild type (A56, mating type +) increase their b-carotene content as they approach each other and form a line of black zygospores. Right. A carotene-overproducer carS mutant (M1, mating type +) increases its b-carotene content and modifies its hyphae for sexual contact in response to the wild type of opposite sex (NRRL1555, mating type ), but fails to induce any effect on the wild type; the interaction remains incomplete and no zygospores are formed.
Fig. 2. A physical and linkage map of genes carB, carRA, and carS and the molecular markers designated by ALID numbers (Table S2). Progeny from a cross between A914 [madI lysA ()] and UBC21 (+) were scored for phototropic defect (madI), lysine auxotrophy (lysA), and the presence of a number of SNPs identified in the genome of UBC21 when compared with the reference strain NRRL1555. (A) The two contigs are separated by a gap in the genome sequence. The numbers in the segments are the number of recombinants in 45 progeny from the cross of strain A914 and UBC21. Gene madI has not been located, but is closely linked with ALID0403. (B) The presence of the A914 allele (A, yellow shading) or the UBC21 allele (B, green shading) for each of the 45 progeny was determined by phenotype or PCR-RFLP. Progeny numbers that were recombinant for the markers located on the chromosome encoding CarS are shaded blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 Mutations and b-carotene production in carS mutants.
a b c
Strain
carS allele
NRRL1555 C115 S178 S100 M1 S324 S303
Wild type carS42 carS153 carS98 carS43 carS180 carS179
Nucleotide change (position)a
CAC to CGC (326) GAG to AAG (535) CCT to TCT (735) TCG to TTG (1438) CAG to TAG (440) CAG to TAG (936)
Amino acid change (position)b
His to Arg (80) Glu to Lys (150) Pro to Ser (199) Ser to Leu (433) Gln to stop (143) Gln to stop (266)
b-carotene (mg/g dry mass)c Dark
Light
0.08 ± 0.01 1.74 ± 0.10 2.22 ± 0.27 0.77 ± 0.02 3.72 ± 0.08 2.85 ± 0.32 3.76 ± 0.08
0.44 ± 0.02 2.28 ± 0.24 2.72 ± 0.14 1.39 ± 0.06 4.71 ± 0.16 3.19 ± 0.05 3.17 ± 0.14
The number refers to the mutant nucleotide position relative to the initiator ATG. The number refers to the mutant amino acid position. Mean and its standard error in 2–5 experiments. All the strains were grown on minimal agar, except strain S303, which was grown on nutrient agar.
aspartates for binding Fe2+ to the active site (Poliakov et al., 2005; Takahashi et al., 2005) that are conserved in functional oxygenases (Kloer and Schulz, 2006; Walter and Strack, 2011). These key amino acids are located in similar positions in the three enzymes of this family whose three-dimensional crystal structures have been solved (Kiser et al., 2009; Kloer et al., 2005; Messing et al., 2010).
3.2. Phenotype of carS mutants In dark-grown cultures, the carS mutants had up to 50 times more b-carotene than the wild type (Table 1). Continuous illumination increased the carotene content of the wild type about five fold, with an absolute increase of about 0.4 mg/g dry mass. A similar
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Fig. 3. Alignment of the amino acid sequences of the predicted CarS protein and three representative b-carotene-cleaving oxygenases from diverse species. Black boxes indicate amino acids present in the same position in the four proteins. The four histidines and the three glutamate considered essential for activity are marked by asterisks. Arrows mark the amino acid substitution sites and crosses mark the stop codon sites in carS mutant strains. Homo sapiens, b-carotene cleavage oxygenase I (BCOI); Fusarium fujikuroi, b-carotene cleavage oxygenase (CarX) and Arabidopsis thaliana, b-carotene cleavage dioxygenase 7 (CCD7). Protein sequences were aligned using the program ClustalW with default parameters (http://www.ebi.ac.uk/Tools/clustalw/).
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absolute increase was seen in the mutants (average 0.6 mg/g dry mass). Strain S303 (allele carS179) did not exhibit photoinduced carotenogenesis: it had less carotene in the light than in dark (0.6 mg/g dry mass), presumably because of the inevitable photolysis. With this exception, the carS mutations did not impede photoinduction of carotenogenesis. Taking photolysis into account, this phenomenon increased the carotene content of the mutants by about 1.2 mg/g dry mass. The alleles carS179 and carS180 introduce premature stop codons. Four mutants carry amino acid substitutions that involve changes in charge, hydrophobicity, or size. The strongest phenotype was caused by allele carS43, which replaces serine 433 by leucine (Table 1). Other substitutions resulted in leaky mutants, the weakest of which was carS98. Strains with mutations in carS are sterile, but strain S100, which carries the allele carS98, produced some zygospores confirming that the mutant protein has retained some activity, but these zygospores failed to germinate and produce progeny. 3.3. Lack of apocarotenoids in carS mutants CarS is a b-carotene-cleaving oxygenase because the carS mutants lack the apocarotenoids made by the wild types. Compounds from cultured media of wild type and car mutants were fractionated by chromatography and detected by spectroscopy (Fig. 4). All the compounds in wild-type culture media that absorbed at 328-nm were neutral apocarotenoids. Some of them have been identified (Polaino et al., 2012) and others are currently under investigation. Five of the carS alleles led to a total absence of neutral apocarotenoids, and only small amounts were seen in the strain with the leaky allele carS98. The 7-carbon apocarotenoids, (2E,4E)-6-hydroxy-5-methylhexa-2,4-dienoic acid and its 2-methyl isomer (Polaino et al., 2010), were detected in the wild types but not in strains carrying any of the six carS alleles under study. No acid compounds were found in the strain with the carS98 allele, but some were detected in small amounts in the other mutants (Fig. 4B). Particularly deficient were the strains with the premature-stop codon alleles carS179 and carS180. 4. Discussion Since the sexual signals in the Mucorales are apocarotenoids (Austin et al., 1970), their synthesis must include splitting the bcarotene carbon chain. We have identified the Phycomyces gene responsible for that reaction, and it comes as a surprise that the gene, carS, was defined long ago as a complementation group of carotene-overaccumulating mutants (Murillo and Cerdá-Olmedo, 1976). The identification rests on the absence of apocarotenoids in the mutants, on the similarity of the CarS sequence to those of well-known b-carotene-cleaving oxygenases, and activity of the CarS protein when expressed in E. coli cells (Medina et al., 2011). There are two groups of mutants unable to produce apocarotenoid signals: those that make no b-carotene (carB and carR), mutants that cannot split it (carS) and mutants that presumably act later in the pathway (Sutter et al., 1996). The sexual phenotype of carB, carR, and carS mutants is the same: they respond to their partners, but do not stimulate them. Since this is true independently of which sex is mutated and no strain stimulates itself, each sex must have its own apocarotenoid signal(s) to send to the other. These signals induce two responses, zygophore formation and increased b-carotene content (sexual carotenogenesis) that can be easily uncoupled (Kuzina and Cerdá-Olmedo, 2006). All carS mutants tested have an increased b-carotene content and a defective b-carotene-cleaving activity. The first could be a
mere consequence of the second, because a metabolite would accumulate if not processed. Many observations support the existence of a feedback regulation in Phycomyces that mediates a strong activation of the carotene pathway by carS mutations, by any genetic or chemical interruption of the pathway, and by retinol, b-ionone, and other b-ring-containing chemicals (Bejarano and Cerdá-Olmedo, 1989; Bejarano et al., 1988; Eslava et al., 1974; Polaino et al., 2010). Critical results supporting a feedback regulation were obtained in cultures with radioactive mevalonate, a precursor of all terpenoids, including b-carotene. In the carS mutant, the total radioactivity in b-carotene was much higher and the specific radioactivity much lower than in the wild type; in both the absolute radioactivity did not diminish appreciably when the mycelia were transferred to non-radioactive media (Bejarano and Cerda-Olmedo, 1992; Kuzina et al., 2006; Murillo et al., 1981). The CarS protein is unlikely to have a regulatory role independent of its enzymatic activity, because otherwise one would expect b-carotene-overproducing carS mutants with normal enzyme activity. The effector that keeps low the carotene content in the wild type should be one of its apocarotenoid products, and an early one containing a b-ring, because the carS mutant phenotype has not been found in mutants of other genes. Two mutants are known that show little increase in b-carotene content in the presence of retinol when compared to wild type: one occurs in the carA domain of gene carRA and the other in another gene, carI (Arrach et al., 2001; Bejarano et al., 1988; Roncero and Cerdá-Olmedo, 1982). These observations are explained if the b-ringapocarotenoid inhibits phytoene synthase, the CarA gene product, which is the first specific enzyme for carotenogenesis, and shuts off the pathway when sufficient b-carotene is available. Retinol and other b-ring-compounds compete for the same target site and thwart that inhibition, presumably because they can bind to CarA and prevent the inhibitory effect of the apocarotenoid. Whether this occurs via an allosteric effect or directly in the site of catalysis of CarA is unclear. This interaction would have to rely on the subtle differences in structures between b-ring-compounds, such as to enable a subset to inhibit enzyme activity and another subset to enhance activity. Exploring this mechanism further will require the identification of the predicted b-ring-apocarotenoid inhibitor, crystal structure information for wild type and mutant forms of CarA bound to these molecules, and comparison of the affinity of those molecules with purified CarA. In keeping with this mechanism, the transcription of the structural genes for carotenogenesis is not modified in a carS mutant (Almeida and Cerdá-Olmedo, 2008) and a cell-free extract of a carS mutant produces about ten times more b-carotene than a parallel extract of the wild type (Salgado et al., 1991). The feed-back regulation of carotenogenesis is independent of and synergic with the sexual activation (Murillo et al., 1978), and we propose that both are mediated by different apocarotenoids produced under different circumstances. There must be b-carotene-cleaving enzymes in other heterothallic Mucorales that use apocarotenoids for sexual signaling. The genomes of Rhizopus oryzae (Ma et al., 2009) and M. circinelloides each contain four sequences related to that of carS. One of them at least should code for a b-carotene-cleaving oxygenase; the closest to carS is sequence RO3G_03330.3 of R. oryzae, and the next is sequence 146755 of M. circinelloides. The same level of similarity is found in the gene called tsp3 in B. trispora, whose product could be a b-carotene-cleaving oxygenase (Burmester et al., 2007). The five Phycomyces genes for putative b-carotenecleaving enzymes are expressed in vegetative mycelia, and carS is induced during mating as expected (Medina et al., 2011). Expression of carS in b-carotene-producing E. coli cells led to formation of b-apo-120 -carotenal, a 25-carbon molecule derived from bcarotene (Medina et al., 2011). This result further confirms the b-carotene-cleaving oxygenase activity predicted for CarS.
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Fig. 4. Fractionation of apocarotenoids. (A) Absorption at 328 nm versus retention time in HPLC chromatograms of neutral extracts of culture media of wild-type and car mutant strains grown for 5 days. The relevant genotypes are indicated. (B) The same for acid extracts, showing absorption at 280 nm. (C) Absorption spectra of the main apocarotenoids of the wild type strain NRRL1555. The values given are the wavelength with maximum absorption, in nm, and the retention time, in min. The compounds are named, following Polaino et al. (2012), according to the chemical group (T, trisporoids; C, cyclofarnesoids; M methylhexanoids, X; unknown), followed by ‘‘n’’ for neutral compounds and ‘‘a’’ for acid compounds, and the peak chromatographic retention time in tens of seconds. To compensate for small variations in loading time the retention times were corrected linearly so that the retention time of compound Cn115 (apotrisporin E) is 18.9 min and that of Xa159 (an acid compound that is not an apocarotenoid but was present in all the acid extracts) is 26.5 min. Values given are the average and standard deviation in 2–6 measurements from 1–3 independent experiments.
Our findings on the carS gene of Phycomyces should not be assumed to apply fully to homologous genes in other Mucorales. The carotene overaccumulating mutants isolated in B. trispora (Mehta and Cerdá-Olmedo, 1995) and in M. circinelloides (Navarro et al., 2000) show only modest increases in carotene content and there are no signs of a feedback control. The b-carotene-cleaving activity in these fungi is likely to be used to produce mating signals but probably not a regulator of carotenogenesis during vegetative growth.
Acknowledgments We thank A. Miyazaki for providing strains, and S. Chaudhary and D. Pérez de Camino for technical help. The Phycomyces genome sequence was provided by the US Department of Energy Joint Genome Institute (Office of Science of the Department of Energy, DE-AC02-05CH11231). This research was funded by the US National Science Foundation (MCB-0920581), by Spanish grants (BIO2009-12486, AGL2005-08081, J. Andalucía P08-CVI-03901,
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P09-CVI-5027 and CVI 910, J. Castilla-León GR64), which are supported by the European Regional Development Fund, and a loan from the Educafin Program, México. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2012.03.002. References Ainsworth, G.C., 1976. Introduction to the History of Mycology. Cambridge University Press, Cambridge. Almeida, E.R., Cerdá-Olmedo, E., 2008. Gene expression in the regulation of carotene biosynthesis in Phycomyces. Curr. Genet. 53, 129–137. Aragón, C.M., Murillo, F.J., de la Guardia, M.D., Cerdá-Olmedo, E., 1976. An enzyme complex for the dehydrogenation of phytoene in Phycomyces. Eur. J. Biochem. 63, 71–75. Arrach, N., Fernández-Martín, R., Cerdá-Olmedo, E., Avalos, J., 2001. A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces. Proc. Natl. Acad. Sci. USA 98, 1687–1692. 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Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi
A gene for carotene cleavage required for pheromone biosynthesis and carotene regulation in the fungus Phycomyces blakesleeanus Víctor G. Tagua a,1, Humberto R. Medina a,1, Raúl Martín-Domínguez b, Arturo P. Eslava b, Luis M. Corrochano a,⇑, Enrique Cerdá-Olmedo a,⇑, Alexander Idnurm c,⇑ a b c
Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41080 Sevilla, Spain Área de Genética, Departamento de Microbiología y Genética, Universidad de Salamanca, Edificio Departamental, 37007 Salamanca, Spain Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri–Kansas City, Kansas City, MO 64110, United States
a r t i c l e
i n f o
Article history: Received 9 December 2011 Accepted 8 March 2012 Available online 21 March 2012 Keywords: Apocarotenoid Beta-carotene Mucoromycotina Pheromone biosynthesis Zygomycete
a b s t r a c t Mating and sexual development in fungi are controlled by molecular mechanisms that are specific for each fungal group. Mating in Phycomyces blakesleeanus and other Mucorales requires pheromones derived from b-carotene. Phycomyces mutants in gene carS accumulate large amounts of b-carotene but do not enter the sexual process. We show that carS encodes a b-carotene-cleaving oxygenase that catalyzes the first step in the biosynthesis of a variety of apocarotenoids, including those that act as pheromones. Therefore carS mutants cannot stimulate their sexual partners, although they respond to them. CarS catalyzes the biosynthesis of a b-ring-containing apocarotenoid that inhibits the activity of the carotenogenic enzyme complex in vegetative cells and provides a feedback regulation for the b-carotene pathway. The carS gene product is a keystone in carotenogenesis and in sexual reproduction. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Phycomyces blakesleeanus is a saprophytic fungus used in many studies of physiology, environmental sensing, genetics and metabolism (Bergman et al., 1969; Cerdá-Olmedo, 2001; Cerdá-Olmedo and Lipson, 1987). Phycomyces and other Mucorales were the first organisms, outside Animals and Plants, known to reproduce sexually (Ainsworth, 1976; Blakeslee, 1904; Idnurm, 2011), but the study of their sexual processes is not as advanced as in the Ascomycete and Basidiomycete fungi (Lee et al., 2010) and other microorganisms. The heterothallic Mucorales are distinctive in the existence of two sexes that look identical and behave symmetrically (isogamy). The two sexes, called (+) and (), are determined by two allelic but dissimilar DNA segments carrying genes sexP and sexM, respectively, whose products are HMG-domain transcription factors (Gryganskyi et al., 2010; Idnurm et al., 2008; Lee et al., 2008; Li et al., 2011). Sexual interaction in Phycomyces and other Mucorales begins with morphological modification of hyphal tips into zygophores and involves an exchange of diffusible signals, the first pheromones discovered in any organism (Burgeff, 1924). These signals are apocarotenoids, derived from fragments of the yellow pigment ⇑ Corresponding authors. E-mail addresses: [email protected] (L.M. (E. Cerdá-Olmedo), [email protected] (A. Idnurm). 1 These authors contributed equally to this work.
Corrochano),
1087-1845/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2012.03.002
[email protected]
b-carotene (Austin et al., 1970; Caglioti et al., 1966; Schimek and Wöstemeyer, 2009), in contrast to the modified peptides used by Ascomycetes and Basidiomycetes (reviewed by Jones and Bennett, 2011). Phycomyces mycelia accumulate all-trans b-carotene synthesized by a carotenogenic enzyme complex (Aragón et al., 1976; Candau et al., 1991; De la Guardia et al., 1971; Sanz et al., 2002) composed of at least seven protein molecules that carry out three different catalytic activities and are the products of two genes, carB and carRA (Arrach et al., 2001; Ruiz-Hidalgo et al., 1997). The b-carotene content of Phycomyces is enhanced by blue light, sexual interaction and many chemicals, and is modified by mutations in various genes (Cerdá-Olmedo, 2001). Blue light is perceived by a photoreceptor and transcription factor complex (Idnurm et al., 2006; Sanz et al., 2009) and stimulates the biosynthesis of b-carotene through the transcription of the carB and carRA genes (Almeida and Cerdá-Olmedo, 2008; Ruiz-Hidalgo et al., 1997; Sanz et al., 2010). Apocarotenoids also increase the carotene content by increasing the transcription of the structural genes during mating (Almeida and Cerdá-Olmedo, 2008), in a separate response from zygophore formation (Kuzina and Cerdá-Olmedo, 2006). A common mechanism mediates the activation of the b-carotene pathway by mutational or chemical inhibition of the carotenogenic enzymes, by retinol or b-ionone added to the medium, and by mutation of carS (Bejarano and Cerdá-Olmedo, 1989; Bejarano et al., 1988; Eslava et al., 1974; Murillo and Cerdá-Olmedo, 1976).
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Apocarotenoids of 18 and 15 carbon atoms have been isolated from the cultures of Phycomyces and other Mucorales. The recent identification of (2E,4E)-6-hydroxy-5-methylhexa-2,4-dienoic acid and other 7-carbon compounds as apocarotenoids (Barrero et al., 2011; Polaino et al., 2010) has shown that the 40-carbon bcarotene molecule is split into three fragments of 18, 7 and 15 carbon atoms, heads of the respective three families of apocarotenoids, the trisporoids, the methylhexanoids, and the cyclofarnesoids, that include those that act as pheromones. Outside of the Dikarya fungi, no gene specific to pheromone biosynthesis has been identified, that is, as mutated in a strain with impaired pheromone production. Here we identify the sequence of gene carS, we show that the carS gene product is a b-carotenecleaving oxygenase responsible for the biosynthesis of apocarotenoids in Phycomyces, and we clarify its role in the regulation of b-carotene production and pheromone biosynthesis. 2. Materials and methods 2.1. Strains and growth conditions Strain NRRL1555, the standard wild type, and other strains of Phycomyces blakesleeanus Bgff. (Mucoromycotina, Mucorales), described in Table S1, were grown at 22 °C in the dark on minimal agar plates inoculated with 104 heat-activated spores. Standard methods for this organism (Cerdá-Olmedo and Lipson, 1987) were followed, unless otherwise stated. Eschericha coli DH5-alpha was used for cloning plasmids. 2.2. Crosses and sexual development The zygospores from the crosses between strains A914 and UBC21, as established on V8 juice agar, germinated and produced germsporangia in about 2 months. A total of 45 progeny colonies, each from a separate germsporangium, was scored for auxotrophy (lysA), phototropism (madI), and restriction fragment polymorphisms after PCR (PCR–RFLP). Sequences for primers for PCR–RFLPs are provided in Table S2. SNPs that change target sites for restriction enzymes were identified after comparison of the genomic sequence of the () wild-type strain NRRL1555 and the Illumina genome sequence of the (+) wild-type strain UBC21. The position of SNPs along the draft of the P. blakesleeanus NRRL1555 genome sequence can be obtained from the JGI web page (http://genome. jgi-psf.org/Phybl1/Phybl1.home.html). The sexual development of the carS mutants was assayed after plating each of them and strain A56 at opposite ends of a potato agar plate. The plates were incubated 2 days at 22 °C and 5–7 days at 16 °C before inspection. 2.3. Sequencing carS alleles Phycomyces genomic DNA was used to amplify and sequence carS alleles in carS mutant strains. PCRs were performed with total genomic DNA, using primers listed in Table S3. PCR products were purified and sequenced by the chain-termination method. The sequence of carS is deposited in GenBank (accession GU339225). 2.4. Analysis of b-carotene and apocarotenoids Cultures were grown for 2 days and then exposed to light at 22 °C for 1 day or kept in the dark as a control. For light exposure the Petri dishes were incubated on a white surface under a set of four fluorescent lamps (Philips TLD 36 W/865, 4 W/m2 white light) placed 75 cm away. After light exposure mycelia were collected, frozen in liquid nitrogen, and lyophilized. Mycelia (about 100 mg
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each) were ground in a mortar with sea sand and a pestle, and extracted with petroleum ether (b.p. 40–60 °C). The extracts were centrifuged (8 min at 4000g), and their b-carotene content was calculated from their absorbance at 453 nm and the extinction coefficient (1 mg/L, 1 cm) = 259.2. The apocarotenoids were extracted and fractionated by reverse-phase HPLC as described (Polaino et al., 2012).
3. Results 3.1. Localization and identification of gene carS Phycomyces mutants in gene carS have a complex phenotype (Fig. 1): they overaccumulate b-carotene and react to strains of opposite sex with additional carotene production and changes in hyphal morphology, but fail to stimulate their partners and complete the sexual cycle (Kuzina et al., 2006; Salgado et al., 1989; Sutter, 1975). The structural genes for carotenogenesis carB and carRA are contiguous (Arrach et al., 2001) on contig 6 of the genome database (release version 1.1). They are genetically linked to carS at about 35 cM (Roncero and Cerdá-Olmedo, 1982) and to madI, a gene required for the phototropism of the fruiting body, at about 27 cM (Campuzano et al., 1995). Thus carS and madI may be linked, and this linkage could help identify carS. A cross was established between strains A914, genotype lysA madI (), and UBC21, a (+) wild type. The auxotrophy and the defective phototropism are easy to test. The two markers segregated independently (14 wild type, 10 lysA, 11 madI, and 10 lysA madI, in 45 progeny) confirming the lack of linkage (Eslava and Alvarez, 1996). When the same progeny were tested for the molecular markers available in contig 6 (Fig. 2), it was found that gene madI lies outside this contig, but not far from one of its ends. In order to identify additional DNA sequence linked to contig 6, the Phycomyces genome was analyzed using BLASTn with the 916-bp sequence at the end of contig 6. The rationale for this approach was based on the hypotheses that (i) contig assembly may be blocked by adjacent repeated DNA sequences and that regions that matched may be on an adjacent contig, and (ii) that repetitive elements can be chromosome-specific. One of the highest regions of similarity was in the middle of contig 32 and a molecular marker in contig 32 was linked to contig 6 (Fig. 2). Two putative genes in contig 32 caught our attention because of their possible relation to carotene metabolism. One of them (JGI ID# 85763) was similar to crgA of Mucor circinelloides, defined by carotene-overproducing mutants (Navarro et al., 2001), but its sequence was the same in a carS strain (M1) and in its parental strain (NRRL1554). The second sequence (ID# 79747) is one of five genes that we were studying independently because of their similarity to well-known carotene-cleaving oxygenases (Medina et al., 2011), and to gene tsp3, encoding a putative carotene oxygenase of Blakeslea trispora (Burmester et al., 2007). The sequence of gene with ID 79747 is carS, because each carS mutant tested (11 strains, six independent alleles) differed from the wild type in a single-base substitution in that sequence (Table 1, Fig. 3). In addition, the carS gene was sequenced in five carotene-overproducing strains (S275, S276, S101, S496, S498) derived from strain C115 by various crosses. All five strains contain the allele carS42 present in the original strain. The carS gene coding region is 2027 bp and contains two introns of 87 and 53 bp as determined by sequencing its cDNA. The predicted CarS protein is 628 amino acid residues in length (Fig. 3). The CarS protein sequence significantly coincides with those of proven b-carotene-cleaving oxygenases from animals, plants and fungi (Fig. 3). It includes four histidines and three glutamates or
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Fig. 1. Strains with mutations in carS are sterile and overproduce b-carotene. Left. Mycelia of the standard wild type (NRRL1555, mating type ) and an isogenic wild type (A56, mating type +) increase their b-carotene content as they approach each other and form a line of black zygospores. Right. A carotene-overproducer carS mutant (M1, mating type +) increases its b-carotene content and modifies its hyphae for sexual contact in response to the wild type of opposite sex (NRRL1555, mating type ), but fails to induce any effect on the wild type; the interaction remains incomplete and no zygospores are formed.
Fig. 2. A physical and linkage map of genes carB, carRA, and carS and the molecular markers designated by ALID numbers (Table S2). Progeny from a cross between A914 [madI lysA ()] and UBC21 (+) were scored for phototropic defect (madI), lysine auxotrophy (lysA), and the presence of a number of SNPs identified in the genome of UBC21 when compared with the reference strain NRRL1555. (A) The two contigs are separated by a gap in the genome sequence. The numbers in the segments are the number of recombinants in 45 progeny from the cross of strain A914 and UBC21. Gene madI has not been located, but is closely linked with ALID0403. (B) The presence of the A914 allele (A, yellow shading) or the UBC21 allele (B, green shading) for each of the 45 progeny was determined by phenotype or PCR-RFLP. Progeny numbers that were recombinant for the markers located on the chromosome encoding CarS are shaded blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 Mutations and b-carotene production in carS mutants.
a b c
Strain
carS allele
NRRL1555 C115 S178 S100 M1 S324 S303
Wild type carS42 carS153 carS98 carS43 carS180 carS179
Nucleotide change (position)a
CAC to CGC (326) GAG to AAG (535) CCT to TCT (735) TCG to TTG (1438) CAG to TAG (440) CAG to TAG (936)
Amino acid change (position)b
His to Arg (80) Glu to Lys (150) Pro to Ser (199) Ser to Leu (433) Gln to stop (143) Gln to stop (266)
b-carotene (mg/g dry mass)c Dark
Light
0.08 ± 0.01 1.74 ± 0.10 2.22 ± 0.27 0.77 ± 0.02 3.72 ± 0.08 2.85 ± 0.32 3.76 ± 0.08
0.44 ± 0.02 2.28 ± 0.24 2.72 ± 0.14 1.39 ± 0.06 4.71 ± 0.16 3.19 ± 0.05 3.17 ± 0.14
The number refers to the mutant nucleotide position relative to the initiator ATG. The number refers to the mutant amino acid position. Mean and its standard error in 2–5 experiments. All the strains were grown on minimal agar, except strain S303, which was grown on nutrient agar.
aspartates for binding Fe2+ to the active site (Poliakov et al., 2005; Takahashi et al., 2005) that are conserved in functional oxygenases (Kloer and Schulz, 2006; Walter and Strack, 2011). These key amino acids are located in similar positions in the three enzymes of this family whose three-dimensional crystal structures have been solved (Kiser et al., 2009; Kloer et al., 2005; Messing et al., 2010).
3.2. Phenotype of carS mutants In dark-grown cultures, the carS mutants had up to 50 times more b-carotene than the wild type (Table 1). Continuous illumination increased the carotene content of the wild type about five fold, with an absolute increase of about 0.4 mg/g dry mass. A similar
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Fig. 3. Alignment of the amino acid sequences of the predicted CarS protein and three representative b-carotene-cleaving oxygenases from diverse species. Black boxes indicate amino acids present in the same position in the four proteins. The four histidines and the three glutamate considered essential for activity are marked by asterisks. Arrows mark the amino acid substitution sites and crosses mark the stop codon sites in carS mutant strains. Homo sapiens, b-carotene cleavage oxygenase I (BCOI); Fusarium fujikuroi, b-carotene cleavage oxygenase (CarX) and Arabidopsis thaliana, b-carotene cleavage dioxygenase 7 (CCD7). Protein sequences were aligned using the program ClustalW with default parameters (http://www.ebi.ac.uk/Tools/clustalw/).
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absolute increase was seen in the mutants (average 0.6 mg/g dry mass). Strain S303 (allele carS179) did not exhibit photoinduced carotenogenesis: it had less carotene in the light than in dark (0.6 mg/g dry mass), presumably because of the inevitable photolysis. With this exception, the carS mutations did not impede photoinduction of carotenogenesis. Taking photolysis into account, this phenomenon increased the carotene content of the mutants by about 1.2 mg/g dry mass. The alleles carS179 and carS180 introduce premature stop codons. Four mutants carry amino acid substitutions that involve changes in charge, hydrophobicity, or size. The strongest phenotype was caused by allele carS43, which replaces serine 433 by leucine (Table 1). Other substitutions resulted in leaky mutants, the weakest of which was carS98. Strains with mutations in carS are sterile, but strain S100, which carries the allele carS98, produced some zygospores confirming that the mutant protein has retained some activity, but these zygospores failed to germinate and produce progeny. 3.3. Lack of apocarotenoids in carS mutants CarS is a b-carotene-cleaving oxygenase because the carS mutants lack the apocarotenoids made by the wild types. Compounds from cultured media of wild type and car mutants were fractionated by chromatography and detected by spectroscopy (Fig. 4). All the compounds in wild-type culture media that absorbed at 328-nm were neutral apocarotenoids. Some of them have been identified (Polaino et al., 2012) and others are currently under investigation. Five of the carS alleles led to a total absence of neutral apocarotenoids, and only small amounts were seen in the strain with the leaky allele carS98. The 7-carbon apocarotenoids, (2E,4E)-6-hydroxy-5-methylhexa-2,4-dienoic acid and its 2-methyl isomer (Polaino et al., 2010), were detected in the wild types but not in strains carrying any of the six carS alleles under study. No acid compounds were found in the strain with the carS98 allele, but some were detected in small amounts in the other mutants (Fig. 4B). Particularly deficient were the strains with the premature-stop codon alleles carS179 and carS180. 4. Discussion Since the sexual signals in the Mucorales are apocarotenoids (Austin et al., 1970), their synthesis must include splitting the bcarotene carbon chain. We have identified the Phycomyces gene responsible for that reaction, and it comes as a surprise that the gene, carS, was defined long ago as a complementation group of carotene-overaccumulating mutants (Murillo and Cerdá-Olmedo, 1976). The identification rests on the absence of apocarotenoids in the mutants, on the similarity of the CarS sequence to those of well-known b-carotene-cleaving oxygenases, and activity of the CarS protein when expressed in E. coli cells (Medina et al., 2011). There are two groups of mutants unable to produce apocarotenoid signals: those that make no b-carotene (carB and carR), mutants that cannot split it (carS) and mutants that presumably act later in the pathway (Sutter et al., 1996). The sexual phenotype of carB, carR, and carS mutants is the same: they respond to their partners, but do not stimulate them. Since this is true independently of which sex is mutated and no strain stimulates itself, each sex must have its own apocarotenoid signal(s) to send to the other. These signals induce two responses, zygophore formation and increased b-carotene content (sexual carotenogenesis) that can be easily uncoupled (Kuzina and Cerdá-Olmedo, 2006). All carS mutants tested have an increased b-carotene content and a defective b-carotene-cleaving activity. The first could be a
mere consequence of the second, because a metabolite would accumulate if not processed. Many observations support the existence of a feedback regulation in Phycomyces that mediates a strong activation of the carotene pathway by carS mutations, by any genetic or chemical interruption of the pathway, and by retinol, b-ionone, and other b-ring-containing chemicals (Bejarano and Cerdá-Olmedo, 1989; Bejarano et al., 1988; Eslava et al., 1974; Polaino et al., 2010). Critical results supporting a feedback regulation were obtained in cultures with radioactive mevalonate, a precursor of all terpenoids, including b-carotene. In the carS mutant, the total radioactivity in b-carotene was much higher and the specific radioactivity much lower than in the wild type; in both the absolute radioactivity did not diminish appreciably when the mycelia were transferred to non-radioactive media (Bejarano and Cerda-Olmedo, 1992; Kuzina et al., 2006; Murillo et al., 1981). The CarS protein is unlikely to have a regulatory role independent of its enzymatic activity, because otherwise one would expect b-carotene-overproducing carS mutants with normal enzyme activity. The effector that keeps low the carotene content in the wild type should be one of its apocarotenoid products, and an early one containing a b-ring, because the carS mutant phenotype has not been found in mutants of other genes. Two mutants are known that show little increase in b-carotene content in the presence of retinol when compared to wild type: one occurs in the carA domain of gene carRA and the other in another gene, carI (Arrach et al., 2001; Bejarano et al., 1988; Roncero and Cerdá-Olmedo, 1982). These observations are explained if the b-ringapocarotenoid inhibits phytoene synthase, the CarA gene product, which is the first specific enzyme for carotenogenesis, and shuts off the pathway when sufficient b-carotene is available. Retinol and other b-ring-compounds compete for the same target site and thwart that inhibition, presumably because they can bind to CarA and prevent the inhibitory effect of the apocarotenoid. Whether this occurs via an allosteric effect or directly in the site of catalysis of CarA is unclear. This interaction would have to rely on the subtle differences in structures between b-ring-compounds, such as to enable a subset to inhibit enzyme activity and another subset to enhance activity. Exploring this mechanism further will require the identification of the predicted b-ring-apocarotenoid inhibitor, crystal structure information for wild type and mutant forms of CarA bound to these molecules, and comparison of the affinity of those molecules with purified CarA. In keeping with this mechanism, the transcription of the structural genes for carotenogenesis is not modified in a carS mutant (Almeida and Cerdá-Olmedo, 2008) and a cell-free extract of a carS mutant produces about ten times more b-carotene than a parallel extract of the wild type (Salgado et al., 1991). The feed-back regulation of carotenogenesis is independent of and synergic with the sexual activation (Murillo et al., 1978), and we propose that both are mediated by different apocarotenoids produced under different circumstances. There must be b-carotene-cleaving enzymes in other heterothallic Mucorales that use apocarotenoids for sexual signaling. The genomes of Rhizopus oryzae (Ma et al., 2009) and M. circinelloides each contain four sequences related to that of carS. One of them at least should code for a b-carotene-cleaving oxygenase; the closest to carS is sequence RO3G_03330.3 of R. oryzae, and the next is sequence 146755 of M. circinelloides. The same level of similarity is found in the gene called tsp3 in B. trispora, whose product could be a b-carotene-cleaving oxygenase (Burmester et al., 2007). The five Phycomyces genes for putative b-carotenecleaving enzymes are expressed in vegetative mycelia, and carS is induced during mating as expected (Medina et al., 2011). Expression of carS in b-carotene-producing E. coli cells led to formation of b-apo-120 -carotenal, a 25-carbon molecule derived from bcarotene (Medina et al., 2011). This result further confirms the b-carotene-cleaving oxygenase activity predicted for CarS.
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Fig. 4. Fractionation of apocarotenoids. (A) Absorption at 328 nm versus retention time in HPLC chromatograms of neutral extracts of culture media of wild-type and car mutant strains grown for 5 days. The relevant genotypes are indicated. (B) The same for acid extracts, showing absorption at 280 nm. (C) Absorption spectra of the main apocarotenoids of the wild type strain NRRL1555. The values given are the wavelength with maximum absorption, in nm, and the retention time, in min. The compounds are named, following Polaino et al. (2012), according to the chemical group (T, trisporoids; C, cyclofarnesoids; M methylhexanoids, X; unknown), followed by ‘‘n’’ for neutral compounds and ‘‘a’’ for acid compounds, and the peak chromatographic retention time in tens of seconds. To compensate for small variations in loading time the retention times were corrected linearly so that the retention time of compound Cn115 (apotrisporin E) is 18.9 min and that of Xa159 (an acid compound that is not an apocarotenoid but was present in all the acid extracts) is 26.5 min. Values given are the average and standard deviation in 2–6 measurements from 1–3 independent experiments.
Our findings on the carS gene of Phycomyces should not be assumed to apply fully to homologous genes in other Mucorales. The carotene overaccumulating mutants isolated in B. trispora (Mehta and Cerdá-Olmedo, 1995) and in M. circinelloides (Navarro et al., 2000) show only modest increases in carotene content and there are no signs of a feedback control. The b-carotene-cleaving activity in these fungi is likely to be used to produce mating signals but probably not a regulator of carotenogenesis during vegetative growth.
Acknowledgments We thank A. Miyazaki for providing strains, and S. Chaudhary and D. Pérez de Camino for technical help. The Phycomyces genome sequence was provided by the US Department of Energy Joint Genome Institute (Office of Science of the Department of Energy, DE-AC02-05CH11231). This research was funded by the US National Science Foundation (MCB-0920581), by Spanish grants (BIO2009-12486, AGL2005-08081, J. Andalucía P08-CVI-03901,
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