The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production
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- Ireland, C., Peekhaus, N., Lu, P. et al. Appl Microbiol Biotechnol (2008) 79: 451. doi:10.1007/s00253-008-1440-3
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Nodulisporic acid A (NAA), an insecticidal indole diterpene, is produced by the fungus Nodulisporium sp. Since indole-3-glycerolphosphate is the precursor of the indole moiety of NAA, it is suggested that the activity of tryptophan synthetase may play a role in NAA biosynthesis. To investigate this hypothesis, the tryptophan synthetase gene TRP1 of Nodulisporium sp. was cloned and characterized. The gene consists of three introns of 146, 68, and 57 bp. The four exons encode a protein of 712 amino acids, the sequence of which is highly homologous to that of other fungal tryptophan synthetase proteins. The transcription initiation site was mapped 66 bp upstream to the ATG, and the polyA tail attachment site is 169 bp downstream to the translation stop codon. Replacement of the N-terminal half of the gene with a hygromycin selection marker yielded mutants with the tryptophan auxotroph/hygromycin-resistance (trp−/hyr) phenotype. The TRP1 mutants required a high concentration of tryptophan supplement in solid medium (10 mM) to sustain minimal growth and failed to produce NAA in the production medium (FFL-CAM) supplemented with high concentrations of tryptophan.
KeywordsTrytophan synthetaseNodulisporium spNodulisporic acid A
Nodulisporic acid A (NAA), isolated from Nodulisporium sp. ATCC74245 (Ondeyka et al. 1997; Ostlind et al. 1997), has potent oral anti-flea activity in dogs and lacks overt mammalian toxicity (Shoop et al. 2001). Its mode of action is by activating a glutamate-gated chloride channel in insects (Smith et al. 2000). Nodulisporium sp. is an anamorphic, endophytic fungus most commonly isolated from woody plants (Polishook et al. 2001). The teleomorph of the fungus is unknown, but morphological and rDNA analysis suggest that it is a Hypoxylon species, close to Hypoxylon fendleri (Monaghan et al. 1995; Polishook et al. 2001; Rodrigues and Samuels 1990).
In this report, we describe the cloning, characterization, and gene disruption of the tryptophan synthetase gene TRP1 from Nodulisporium sp. and its effect on the production of NAA. During the course of this study, we also optimized a fermentation medium for NAA production by Nodulisporium sp. ATCC74245. Experiments showed that the TRP1 mutants needed a high concentration of tryptophan supplement in solid medium (10 mM) to sustain minimal growth and failed to produce NAA in fermentation media supplemented with high concentrations of tryptophan.
Materials and methods
Strains, media, and reagents
Escherichia coli strains DH5α and JM109 were from Gibco BRL (Gaithersburg, MD, USA) and Promega (Madison, WI, USA), respectively. E. coli strains XL1-blue-MR, XL2-blue, and XL-10 Gold were from Stratagene (La Jolla, CA, USA). Nodulisporium sp. ATCC74245 transformation medium, R2YE with hygromycin (Hy, 500 μg/ml) and cyclosporin (1.0 μg/ml), was prepared as described previously (Fulton et al. 1999). Potato dextrose agar (PDA) and PDA + 10 mM tryptophan media were used for scoring tryptophan auxotrophic mutants of Nodulisporium sp. Restriction endonucleases and DNA-modifying enzymes were from Gibco BRL. Chemicals were from Sigma (St. Louis, MO, USA) and Fisher Scientific (Pittsburgh, PA, USA).
Oligonucleotides used in this study
Sequence 5′ to 3′
PCR TRP3 probe
PCR TRP3 probe
PCR TRP3 probe
PCR TRP3 probe
PCR TRP3 probe
Nucleotide and amino acid sequence analysis
The nucleotide sequence of a 5.3-kb EcoRI DNA fragment, which contained the tryptophan synthetase gene and flanking DNA of Nodulisporium sp., was determined by a random partial Sau3AI DNA fragments sequencing procedure. Sequencing was performed using an ABI 377 automated sequencer (PE Biosystems, Foster City, CA, USA). DNA sequence fragments were assembled into contigs using Sequencher version 4.0.2 (Gene Codes, Ann Arbor, MI, USA). Multiple sequence alignment was performed using the ClustalX version 1.8.
RNA and cDNA procedures
For total RNA isolation, mycelia of Nodulisporium sp. ATCC74245 grown in acetate medium (per liter: glucose, 0.5 g; Difco yeast extract, 1 g; potassium acetate, 10 g) were harvested using Miracloth (Calbiochem, La Jolla, CA, USA). Mycelia were then collected, frozen in liquid nitrogen, and dried in a vacuum centrifuge (SpeedVac, SVC100, Framingdale, NY, USA). The dried mycelia were ground to powder and the Tri Reagent RNA isolation kit (MRC, Cincinnati, OH, USA) was used to isolate the RNA. RNA was precipitated by addition of isopropanol and solubilized in water. RNA Reliant Gels (Promega) were used to quantify and visualize RNA in addition to optical density determination at 260 nm. The Promega PolyATract mRNA Isolation system was used to isolate mRNA. Complementary DNA (cDNA) fragments were synthesized using the Perkin-Elmer GeneAmp Thermostable rTth reverse transcriptase RNA PCR kit (RT-PCR). Introns were identified by comparison of the genomic and cDNA sequences of the gene. The 5′ rapid amplification of cDNA ends (RACE) kit Version 2.0 (Life Technologies, Rockville, MD, USA) was used to determine the 5′ end of the message. The two primers used in the 5′ RACE experiment were ts1r and ts3r. The polyA attachment site of the gene was mapped by the 3′-RACE System for Rapid Amplification of cDNA Ends Kit (Life Technologies). The four primers used in the 3′ RACE experiment were ts1f, tsprimer3, and CNP4.
Gene replacement construct
The gene replacement plasmid pTSHyg was constructed as follows: A 5.3 kb DNA fragment containing the tryptophan synthetase gene was generated using PCR primers with BamHI linkers DP1 and DP2 and subcloned into pUC18 (Stratagene) to give pUCNS-trp. The 1.1 kb NcoI fragment, which contains the N-terminal portion of the tryptophan synthetase gene in pUCNS-trp, was then replaced by the 3.5 kb BglII/XbaI Hy-resistant cassette isolated from pMLF2 (An et al. 1996) by blunt-end ligation to give pTSHyg. The gel-purified 7.7 kb BamHI fragment was used in Nodulisporium sp. transformation.
Isolation of gene disruption mutants
Protoplasting and transformation of Nodulisporium sp. ATCC74245 were performed as described (Fulton et al. 1999; Yelton et al. 1984). Transformation plates were incubated at 25°C in darkness. Hy-resistant colonies appeared about 10 days after plating. Hy-resistant transformants were screened for the tryptophan auxotrophic phenotype by plating the transformants on both PDA and PDA + 10 mM tryptophan media.
To prepare inoculum, 50 ml of S3 seed medium in 250-ml unbaffled Erlenmeyer flasks was inoculated with 1 ml of frozen vegetative mycelium. One liter of S3 medium (pH 6.0) contains monosodium glutamate, 10 g; NH4Cl, 3 g; MES, 20 g; K2HPO4, 1 g; MgSO4 7H2O, 0.5 g; glucose, 50 g; CaCO3, 1 g; Amicase, 2 g; and trace elements 1B, 20 ml. One liter of trace elements 1B contains FeSO4·7H2O, 0.5 g; ZnSO4·7H2O, 0.5 g; MnSO4·H2O, 0.1 g; CuSO4·5H2O, 0.05 g; and CoCl2·6H2O, 0.04 g. The flasks were shaken at 220 rpm and at 29°C for 72 h in the dark. For the production of NAA, 250-ml unbaffled Erlenmeyer flasks with 40 ml of NAA production medium (FFL-CAM) were inoculated with 1 ml of homogenized seed inoculum. One liter of FFL-CAM contains monosodium glutamate, 2 g; NH4Cl, 4.6 g; MES, 20 g; K2HPO4, 1 g; MgSO4 7H2O, 0.5 g; glycerol, 80 g; glucose, 74 g; CaCO3, 1 g; amicase, 1 g; L-tryptophan, 1 g; lactic acid (85%), 5 ml; and trace elements 1B, 20 ml. The flasks were shaken at 220 rpm and at 29°C for up to 21 days in the dark.
Fermentation samples in FFL-CAM medium were taken at 7, 10, 14, 17, and 21 days. After pH determination, the contents of the flasks were homogenized. Aliquots of 10 ml homogenate were extracted with 20 ml of mitogen-activated and extracellular signal-regulated kinase. The solvent was evaporated to dryness and resuspended in 1 ml of methanol for high performance liquid chromatography (HPLC) analysis. Production of NAA was measured using an 1100 series HPLC instrument (Agilent Technologies, Waldbronn, Germany) with the following conditions: column, Symmetry ODS C18 (Waters, Milford, MA, USA); solvent mixture, 0.1% Trifluoroacetic acid 35% and acetonitrile 65%; flow rate, 0.9 ml/min; column temperature, 50°C; and detection, 270 nm.
Dry weight determination
Fermentation was carried out as described above and sampled at 7, 10, 14, 17, and 21 days. Samples were homogenized, and 2 ml aliquots were spun at 3,500 rpm for 10 min. Supernatant was saved for the analysis of carbohydrates and tryptophan. Cell pellets were rinsed three times in 5 ml deionized H2O and resuspended in 2 ml deionized H2O by vortexing. Samples were pipetted into tared weigh boats and dried to constant weight in a vacuum oven at 60°C with a vacuum of 20 in. of mercury. Dry weights were determined in duplicate for each sample.
Supernatants prepared as described above were diluted 1:4 with sterile deionized H2O. Diluted samples were filtered through a 0.45 μM filter to remove the particulates. Spectra-Physics System P4000 (Bio-Rad, Hercules, CA, USA) was used for sugar detection using an Aminex HPX-87H HPLC column. The column was run isocratically at 50°C with 0.005 M H2SO4 at a flow rate of 0.7 ml/min, and the carbohydrates were detected with a refractive index detector.
To quantitate residual tryptophan in the medium, fermentation supernatant was first derivatized as previously described (Marfey 1984). The reaction was carried out at 40°C for 1.5 h. The reaction was stopped by addition of 80 μl of 1 M HCl, and methanol was added to bring the final volume to 1 ml. This was assayed using the 1100 series HPLC instrument with the following conditions: column, Symmetry ODS C18, solvent mixture, 0.1% aqueous trifluoroacetic acid 90% and acetonitrile 10%; flow rate, 1.0 ml/min; column temperature, 25°C; and detection, 340 nm.
GenBank accession number
The DNA and amino acid sequences for TRP1 of Nodulisporium sp. has been deposited in the GenBank database with the accession number AY206418.
Cloning the Nodulisporium sp. tryptophan synthetase gene
Comparisons of six fungal tryptophan synthetases and their encoding genes
Protein size (aa)
Identity to TRP1 (%)
A domain to TRP1 (%)
B domain to TRP1 (%)
Connecting region (aa)
RT-PCR message analysis, transcription start site, and polyadenylation site
The cDNA sequence of the gene was determined by RT-PCR using primers designed to generate overlapping cDNA fragments. Comparison of the cDNA and genomic sequences of the genes revealed three introns. The sizes of the three introns are 146, 68, and 57 bp, respectively.
The transcription initiation site of the gene was identified at 66 bp upstream of the expected translation start codon by 5′-RACE experiment. The same site was mapped by primers ts1r and ts3r, located downstream of the first two introns of the gene. The sequences of the two RT-PCR products had both introns 1 and 2 excised indicating the correct cDNA fragments were isolated.
The transcription termination site of the gene was identified at 169 bp downstream of the translation stop codon (TAA) by 3′-RACE experiment. The same site was mapped with four different primers upstream of the translation stop codon. Two of the four primers (RTPCR-2 and ts1f) are located upstream of the third intron. As expected, the sequence of these RT-PCR products had the intron excised. These results indicate that the correct transcription termination site for the gene was mapped.
Based on the sequence alignment with the Neurospora crassa gene and the consensus fungal nucleotide sequence flanking the translation start codon of fungal genes [CA(C/A)(A/C)ATGGC] (Ballance 1991), the ATG start codon for the Nodulisporium sp. tryptophan synthetase gene was predicted (CACCATGGA). The translation termination codon TAA was also predicted based on the sequence alignment with the Neurospora crassa gene.
Disruption of the Nodulisporium sp. tryptophan synthetase gene
Kinetics of NAA production by TRP1− Mutants
The significant amino acid sequence homology between the TRP1 gene and other fungal tryptophan synthetase encoding genes suggests that TRP1 encodes tryptophan synthetase in Nodulisporium sp. The tryptophan auxotrophic phenotype associated with TRP1 disruption mutants further demonstrates that we have cloned the tryptophan synthetase gene from Nodulisporium sp. The transcription termination site of TRP1 was mapped at 169 bp downstream of the last codon, and this falls in the range of untranslated 3′ RNA in Neurospora crassa, which is between 27 and 540 bp (Bruchez et al. 1993). No clear eukaryotic polyadenylation signal (AATAAA or AATAA) was found upstream of the poly(A) tail, but an AATTCA sequence 19 bp upstream of the poly(A) tail could be the polyadenylation signal of TRP1.
The three introns in TRP1 are 146, 68, and 57 bp in size, and this range is within the 52–367 bp range of introns in Neurospora crassa (Radford and Parish, 1997). All three introns have the 5′-XX/GT and 3′-XX/AG splicing consensus junctures, and CT(A/G)A(T/C) consensus lariat sequence can be found in two of the three introns. The amino acid sequences of the six fungal tryptophan synthetase genes are compared in Table 2. The first two introns of TRP1 are located near the 5′ end of the gene. This is similar to the two introns in the TRP-3 of Neurospora crassa, but the third intron in TRP1 is not present in the Neurospora crassa TRP-3 gene (Burns and Yanofsky 1989). In contrast, the trpB gene of A. nidulans contains only one intron (Eckert et al. 2000), the C. cinereus TRP1 open reading frame is disrupted by nine introns (Skrzynia et al. 1989), and there are no introns in either TRP5 of Saccharomyces cerevisiae or the Schizosaccharomyces pombe tryptophan synthetase genes (Zalkin and Yanofsky 1982). Evolution studies suggest that fungal tryptophan synthetase genes were gene fusion products of the separate tryptophan synthetase α and β polypeptides of bacteria (Burns et al. 1990; Burns and Yanofsky 1989). It is not clear whether introns were acquired or lost during the course of tryptophan synthetase gene evolution, since some of the fungal genes contain introns and others do not. Phylogenetic analysis of the six fungal tryptophan synthetase proteins grouped the Nodulisporium sp. TRP1, the A. nidulans trpB, and the Neurospora crassa TRP-3 into one group, the Saccharomyces cerevisiae and the Schizosaccharomyces pombe protein into another group, and the C. cinereus TRP1 was branched as its own group (data not shown). This is not surprising, since Nodulisporium sp., A. nidulans, and Neurospora crassa are all filamentous ascomycetes, Saccharomyces cerevisiae and Schizosaccharomyces pombe are yeasts, and C. cinereus is the only basidiomyceteous fungus in the group. The B domains are more conserved than the A domains in the six fungal tryptophan synthetases (Table 2). DNA sequences that connect the A and B domains share no similarities but are of similar length (ranging from 40 to 69 bp; Table 2). In all six proteins, the connecting regions are flanked by a tyrosine at the N terminus and a proline at the C terminus (data not shown). This finding supports the hypothesis that the connecting region serves as a tether linking the two independent functional domains and that the length of the connecting region is more critical than its sequence (Burns et al. 1990; Burns and Yanofsky 1989). It is interesting that the connecting region in the A. nidulans trpB protein is considerably longer (69 aa) than that of the other five fungal tryptophan synthetase proteins (ranging from 40 to 58 aa in length, Table 2).
The homologous gene replacement rate for TRP1 is about 20%. In contrast, the homologous gene replacement rate for the pks1 gene of Nodulisporium sp. was over 70% (Fulton et al. 1999). The difference in the homologous recombination rate may be due to the length of the homologous DNA flanking the selection marker of the disruption construct. In the TRP1 disruption cassette, the lengths of the homologous flanking DNAs were 1.5 and 2.5 kb, but in the pks1 disruption construct, the flanking DNAs were 3 and 4.6 kb in length (Fulton et al. 1999).
Tryptophan synthetase gene disruption mutants of Nodulisporium sp. were unable to grow on unsupplemented PDA medium but grew on PDA containing 10 mM tryptophan. Deletion of the tryptophan synthetase gene trpB in A. nidulans requires 100 μM for vegetative growth on solid medium and 30 mM for normal spore pigmentation (Eckert et al. 2000). The addition of 50 mM tryptophan in solid medium can induce cleistothecia primordia formation in A. nidulans trpB-deletion strains, but no viable ascospores were produced (Eckert et al. 2000). The requirement of high tryptophan concentration by A. nidulans trpB-deletion strain was suggested to be due to limitation in uptake capacity (Eckert et al. 2000). Tryptophan exhaustion by day 7 in the Nodulisoporium sp. tryptophan synthetase gene disruption mutants indicated that there was no limitation in uptake capacity.
Fermentation medium FFL-CAM was optimized for NAA production by the wild-type strain under various tryptophan concentrations from 2.5 to 50 mM. Tryptophan supplement at 5 mM supported the minimum growth of the mutant strain and had no negative impact on the growth and NAA production in the wild-type strain. The mutant strain grows well at tryptophan concentrations up to 50 mM; however, toxicity was observed in the wild-type strain at such high concentrations (data not shown).
Growth of the mutant in FFL-CAM as determined by dried biomass was similar to the wild type in the first 10 days, but the growth stopped after day 10, while the wild type continued to grow. During the first 10 days, both the wild type and the mutant depleted glucose in the medium. Since Nodulisporium sp. cannot use glycerol as a carbon source for growth (unpublished finding), the carbon source for subsequent growth of the wild type must have come from the stored metabolites of glucose. It was interesting that even though glycerol could not be used for growth, it was metabolized completely by the wild type and over 50% by the mutant. For wild type, as shown in Fig. 3, glycerol utilization occurred concurrently with the production of NAA. In contrast, despite substantial utilization of glycerol, no NAA was made by the mutant. Even at 50 mM tryptophan concentration, no NAA production was observed by the mutant strain (data not shown). It is proposed that glycerol provides the precursors and energy for the synthesis of NAA; however, the role of glycerol in the synthesis of NAA is not well understood. Disruption of TRP1 and exhaustion of tryptophan by day 7 (data not shown) should have increased the precursor of the indole moiety IGP (Byrne et al. 2002) available for the biosynthesis of NAA, so it could not have been limiting. Furthermore, metabolism of glycerol suggests that energy and precursors for mevalonate pathway were also available. A possible explanation is that tryptophan is involved in the regulation of the NAA biosynthetic pathway. It is known that tryptophan is a precursor of ergot alkaloids and induces their biosynthesis (Floss and Mothes 1964; Vining 1970). The target of the action of tryptophan was identified as dimethylallyltryptophan synthetase, the first enzyme of alkaloid biosynthesis (Krupinski et al. 1976). If tryptophan plays a similar role in NAA biosynthesis, its low intra-cellular concentration in the auxotroph may result in the lack of induction of the pathway and therefore NAA production.
The TRP1 mutants of Nodulisporium sp. were readily transformed to prototrophy with TRP1, and therefore, TRP1 can be used as a selectable marker for Nodulisporium sp.
We thank John Ondeyka for the preparation of Fig. 1; Thomas Fulton for use of the Nodulisporium sp. genomic cosmid library, and Maria Losada for assistance in the RNA experiments. We also thank William Strohl, Jan Tkacz, Richard Monaghan, and Thomas Fulton for critically reading the manuscript.