Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA1
Infectious Diseases Section, VA Connecticut Healthcare System, West Haven, CT, USA2
Author for correspondence: Brian Wong. Tel: +1 203 937 3446. Fax: +1 203 937 3476. e-mail: brian.wong{at}yale.edu
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ABSTRACT |
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Keywords: mannitol-1-phosphate dehydrogenase, Cryptococcus neoformans, polyol/alcohol dehydrogenases
Abbreviations: M1Pase, mannitol-1-phosphatase; MPD, mannitol-1-phosphate dehydrogenase
The GenBank accession numbers for the nucleotide sequences for the C. neoformans mannitol-1-phosphate dehydrogenase cDNA and gene are AF175685 and AF186474, respectively.
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INTRODUCTION |
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The findings summarized above suggested that mannitol may function in C. neoformans and other fungi as an intracellular osmolyte and stress protectant and that it may be required for wild-type virulence. However, this hypothesis has not yet been tested directly because the pathway by which C. neoformans synthesizes mannitol is not yet known. In 1980, Hult et al. proposed that several other fungi synthesize and catabolize mannitol via a unidirectional mannitol cycle. In the biosynthetic arm of this pathway, fructose 6-phosphate is reduced to mannitol 1-phosphate by NAD-dependent MPD, after which mannitol-1-phosphatase (M1Pase) dephosphorylates mannitol 1-phosphate to yield mannitol. MPD and M1Pase have been demonstrated in several fungi (Jennings, 1984 ) and the biochemical properties of some fungal MPDs and M1Pases have been described (Jennings, 1984
; Wang & Le Tourneau, 1972
; Kiser & Niehaus, 1981
). However, neither fungal mutants lacking either MPD or M1Pase nor cDNAs or genes encoding fungal MPDs or M1Pases have been described. Therefore, the goals of the present study were (i) to purify and characterize MPD from C. neoformans and (ii) to clone and analyse the corresponding cDNA and gene.
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METHODS |
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Libraries.
A library of C. neoformans H99 cDNAs in the GAL1-regulated yeast expression plasmid pYES2.0 (Invitrogen) was constructed as follows. C. neoformans H99 was grown to mid- to late-exponential phase in YPD, and the cells were harvested by centrifugation and disrupted by passage through a French pressure cell (SLM Instruments) at 40000 p.s.i. (276000 kPa). Total RNA in the cell lysate was isolated using the Bio101 Fast RNA kit. The mRNA was isolated from the total RNA using the Fasttrack 2.0 kit (Invitrogen), and cDNAs were synthesized using oligo(dT)NotI primers and AMV reverse transcriptase from the Copy kit (Invitrogen) according to the manufacturers instructions. EcoRI adapters were added to the size-selected cDNAs (1 kb), and they were cloned unidirectionally into pYES2.0. The final cDNA library consisted of 3·9x106 independent clones with a mean insert size of 1·75 kb, and it was amplified twice in Escherichia coli prior to use. A library of C. neoformans H99 genomic DNA fragments in LambdaZapII (Stratagene) was provided by John Perfect (Duke University, Durham, NC, USA).
Chemicals.
All chemicals, sugar phosphates, sugar alcohols, nucleotide cofactors, and protease inhibitors were from Sigma.
Enzyme assays.
MPD activity was measured by monitoring NADH appearance at 340 nm when the enzyme was added to 0·5 mM mannitol 1-phosphate and 5 mM NAD in 10 mM HEPES (pH 9·0). Fructose-6-phosphate reductase activity was measured by monitoring NADH disappearance after the enzyme was added to 3 mM fructose 6-phosphate and 5 mM NADH in 10 mM HEPES (pH 7·0).
S. cerevisiae strains YPH252 and YPH252 gpd1::leu2 were transformed with plasmids using the lithium acetate method (Sherman et al., 1986
) and the transformants were tested for MPD activity as described above. The S. cerevisiae transformants were also tested for the ability to produce mannitol from glucose by culturing in SC gal-ura and by analysing log-and stationary-phase cells by gas chromatography as previously described (Wong et al., 1989
). Lastly, the transformants were tested for their abilities to tolerate osmotic stress by culturing in YPD supplemented with 0·22·5 M NaCl.
Purification of MPD.
C. neoformans H99 was grown to late-exponential phase in 12 l YPD and the cells were harvested by centrifugation. After harvesting, 240 ml packed cells were suspended to form a paste in 150 ml 50 mM Tris/HCl buffer (pH 7·5) containing 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0·2 mM PMSF, 1 µg leupeptin ml-1 and 1 µg pepstatin ml-1 and the paste was passed twice through a French pressure cell at 40000 p.s.i. (276000 kPa). Cell debris was removed by centrifugation at 16000 g for 30 min, and the supernatant, after centrifugation at 100000 g for 1 h, was used for the further purification. Protamine sulphate solution (2%, w/v; 0·1 vol.) was added, and the suspension was centrifuged at 16000 g for 10 min. The protamine sulphate supernatant was loaded onto a Q12 ion-exchange column (15x68 mm; Bio-Rad) that had been equilibrated with 50 mM Tris/HCl buffer (pH 7·5) using a Bio-Rad Biologic medium-pressure liquid chromatograph. The column was washed with 25 ml 50 mM Tris/HCl buffer (pH 7·5) and bound proteins were eluted with a 0200 mM NaCl gradient (75 ml). Fractions containing MPD activity were pooled, and the buffer was changed to 10 mM HEPES (pH 8·0). The sample was applied to a Matrex blue A gel column (15x100 mm, Millipore), the column was washed with 40 ml 10 mM HEPES (pH 8·0) and the MPD was eluted with 5 ml 0·1 mM NADH. This fraction was concentrated and the buffer was changed to 50 mM Tris/HCl (pH 7·5), 100 mM NaCl. The sample was subjected to gel filtration on a FPLC Superdex 200 HR 10/30 size-exclusion column (Amersham), and the active fractions were collected and concentrated by ultrafiltration; the final sample was stored at 4 °C after addition of 10% (v/v) glycerol and 1 mM DTT. Protein concentrations were determined by the Bradford method, using bovine serum albumin as the standard (Bradford, 1976 ).
Properties of C. neoformans MPD.
SDS-PAGE was performed by the method of Laemmli (1970) and two-dimensional electrophoresis by the method of OFarrell (1975)
. Gels were stained for proteins with Coomassie blue and non-denaturing gels were stained for MPD activity in 10 mM HEPES (pH 9·0), 2 mM NAD, 0·75 mg nitro blue tetrazolium ml-1, 0·125 mg phenazine methosulphate ml-1, and 0·38 mM mannitol 1-phosphate at 37 °C for 5 min.
The native molecular size of MPD was estimated by size-exclusion chromatography with a FPLC Superdex 200 HR 10/30 column that had been equilibrated with 50 mM Tris (pH 7·5), 100 mM NaCl. Sweet potato ß-amylase (200 kDa), yeast alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), bovine erythrocyte carbonic anhydrase (29 kDa) and horse heart cytochrome c (12·4 kDa) were used as molecular mass standards.
The substrate specificities of MPD were assayed by monitoring A340 after the enzyme was added to 5 mM of the polyol, sugar phosphate or polyol phosphate of interest and either 0·5 mM NAD in 10 mM HEPES (pH 9·0) or 0·5 mM NADH in 10 mM HEPES (pH 7·0). Cofactor specificity was assessed by substituting NADP for NAD and NADPH for NADH.
Kinetic constants were determined with 0·2 U purified MPD. The concentrations of mannitol 1-phosphate and NAD were varied from 0·005 to 0·07 mM and 0·01 to 0·2 mM, respectively. The concentration for ethanol varied from 0·5 mM to 3·0 mM. Kinetic constants for the reverse reaction were obtained using fructose 6-phosphate concentrations from 0·02 to 0·5 mM and NADH concentrations ranging from 0·01 to 0·1 mM. Km and Vmax values were calculated from LineweaverBurk plots.
The effects of pH were determined in 10 mM MES buffer at pH 6·0 and 6·5, 10 mM HEPES buffer at pH 7·0, 7·5, 8·0, 8·5 and 9·0, and 10 mM CAPS buffer at pH 9·5 and 10·0. The effects of temperature were determined by incubating the reaction mixture at various temperatures ranging from 25 °C to 65 °C and measuring NADH appearance at 340 nm.
Peptide sequence determination.
N-terminal sequencing of purified C. neoformans MPD and of tryptic peptides that had been purified by reverse-phase HPLC was performed by the Macromolecular Resources Laboratory at the University of Kentucky.
Isolation and analysis of the MPD cDNA and gene.
Standard methods (Sambrook et al., 1989 ) were used to synthesize oligonucleotide primers based on partial peptide sequence data, to amplify DNA by PCR and to prepare [
-32P]dATP-labelled probes. These probes were used to screen the C. neoformans cDNA library by colony hybridization and the C. neoformans genomic DNA library by plaque hybridization. Positive clones were purified, mapped, subcloned and sequenced using standard methods. Multiple sequence alignments were performed by CLUSTAL W (Thompson et al., 1994
).
Southern and Northern hybridizations.
C. neoformans H99 genomic DNA was isolated by the method of Sherman et al. (1986) . Genomic DNA (5 µg per lane) was digested with the restriction enzymes BamHI, HindIII, ClaI, SmaI, SalI, PstI, BglII and XbaI, separated by agarose gel electrophoresis, transferred to a nylon membrane and hybridized with a 1083 bp 32P-labelled MPD cDNA using standard methods (Sambrook et al., 1989
).
Total RNA was isolated by the method of Sherman et al. (1986) from C. neoformans strains H99 and MLP that had been cultured in SC-glucose or SC-mannitol at 30 °C and from C. neoformans H99 that had been cultured in YPD at 25 °C, 30 °C or 37 °C and in YPD containing 0·21·5 M NaCl at 30 °C. RNA (10 µg per lane) was fractionated by electrophoresis on 1% agarose gels, transferred to nylon membranes, and the membranes were hybridized with a 600 bp 32P-labelled partial MPD cDNA, followed by autoradiography. Gel loading was normalized by probing duplicate blots for actin mRNA; the actin probe was amplified from C. neoformans genomic DNA by PCR using primers 5'-TCGCCGCCTTGGTCATTG-3' and 5'-CGTATTCGCTCTTCGCGA-3' (Cox et al., 1995
). Band densities were measured with the EagleEye photodocumentation system and EagleSight software (Stratagene).
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RESULTS |
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The kinetic constants for the NAD-dependent oxidation of mannitol 1-phosphate and for NADH-dependent reduction of fructose 6-phosphate were determined with 0·2 U enzyme. The following constants were derived from the LineweaverBurk plot: Vmax 0·40 µmol mg-1 min-1 and Km 0·055 mM for mannitol 1-phosphate; Vmax 0·17 µmol mg-1 min-1 and Km 0·11 mM for NAD; Vmax 0·31 µmol mg-1 min-1 and Km 0·30 mM for fructose 6-phosphate; Vmax 0·21 µmol mg-1 min-1 and Km 0·099 mM for NADH. The Km and Vmax for ethanol were 0·94 mM and 0·04 mmol mg-1 min-1, respectively.
NAD-dependent oxidation of mannitol 1-phosphate was optimal at pH 9·09·5 and NADH-dependent reduction of fructose 6-phosphate was optimal at pH 7·07·5. Optimal MPD activity was at 37 °C and MPD was stable for 1 week at 4 °C. Addition of CaCl2 (1 mM), NaCl (200 mM), EDTA (5 mM), MgSO4 (5 mM), NiSO4 (5 µM) and ZnSO4 (2 µM) had little effect on MPD activity (<10% difference compared to standard buffer), but addition of MnSO4 (5 mM) increased activity by 220%. However, neither the salts listed above nor EDTA changed the NAD-dependent oxidation of ethanol.
Peptide sequences
Efforts to sequence the N terminus of purified MPD were unsuccessful, which suggested that the N terminus was blocked. Therefore, a tryptic digest of purified MPD was separated by reverse-phase HPLC and three well-resolved tryptic peptides were sequenced. The partial sequences obtained from these three peptides were (i) LSGYTTDGTFSEY, (ii) (K or R)GVFEDLEAG and (iii) (K or R)SLGADAWVDF (Fig. 2.)
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Neither this cDNA sequence nor its deduced protein product was homologous to the nucleotide or deduced peptide sequences of mannitol-1-phosphate dehydrogenases from several bacteria (Brown & Bowles, 1977 ; Fischer et al., 1991
; Novotny et al., 1984
) or the protozoon Eimeria tenella (Schmatz, 1997
). However, a BLAST search of the GenBank and SWISS-PROT databases (Altschul et al., 1997
) revealed that the deduced peptide was 4347% identical to long-chain alcohol/polyol dehydrogenases from several fungi and that it was most similar to alcohol dehydrogenase III from Aspergillus nidulans. The deduced peptide also contained conserved residues for cofactor binding (Wierenga & Hol, 1983
) and conserved residues for two active sites containing zinc (Jornvall et al., 1987
) (Fig. 3
).
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Northern blot analysis
Northern blotting experiments demonstrated the 1·6 kb MPD mRNA in C. neoformans cells grown in SC-glucose, SC-mannitol, or YPD and they showed that MPD mRNA levels were only 1·2-fold higher in cells grown in mannitol than in those grown in glucose. When MPD mRNA levels were examined in C. neoformans cells grown at various salt concentrations, MPD mRNA levels increased as NaCl concentrations were increased from 0 to 0·4 M NaCl and they decreased as NaCl concentrations were increased from 0·6 to 1·5 M (Fig. 5a). In addition, MPD mRNA levels were threefold higher in cells grown at 30 °C or 37 °C than in cells grown at 25 °C (Fig. 5b
). Lastly, the amounts of MPD mRNA were similar in glucose- and mannitol-grown cultures of wild-type C. neoformans H99 and the mannitol-underproducing mutant of C. neoformans MLP (Fig. 5c
).
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DISCUSSION |
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Although the most active substrates for C. neoformans MPD were mannitol 1-phosphate and fructose 6-phosphate, this enzyme also catalysed the interconversion of sorbitol 6-phosphate with glucose 6-phosphate, as do the MPDs of Pyrenochaeta terrestris and Sclerotinia sclerotiorum (Jennings, 1984 ; Wang & Le Tourneau, 1972
). C. neoformans MPD resembled A. niger MPD in that it was exclusively NAD-dependent (Kiser & Niehaus, 1981
). In contrast, E. tenella MPD can utilize NAD or NADP as cofactors (Schmatz, 1997
). The Km and Vmax values of C. neoformans MPD for mannitol 1-phosphate, fructose 6-phosphate, NAD and NADH were similar to those of A. niger MPD (Kiser & Niehaus, 1981
). Unlike the Aspergillus parasiticus enzyme, zinc had no effect on the thermal stability of C. neoformans MPD (Foreman & Niehaus, 1985
). Like most dehydrogenases, the optimal activity for the oxidation of mannitol 1-phosphate was at pH 9·09·5, whereas optimum reduction of fructose 6-phosphate was at pH 7·07·5. C. neoformans MPD was highly active at 37 °C, whereas the optimal temperature for E. tenella MPD was 41 °C (Schmatz, 1997
).
We used partial peptide sequences derived from purified C. neoformans MPD to generate a specific hybridization probe and to clone the C. neoformans MPD cDNA by hybridization. Three independent lines of evidence support the conclusion that the cDNA of interest encodes C. neoformans MPD. First, the molecular size of the deduced product encoded by the cDNA of interest and the subunit molecular size of C. neoformans MPD as determined by denaturing SDS-PAGE were very similar (37·5 kDa and 36 kDa, respectively). Second, all three partial peptide sequences derived from purified C. neoformans MPD (including one that was not used to generate the hybridization probe) were encoded by the cDNA of interest. Third, lysates of S. cerevisiae cells that had been transformed with the cDNA of interest contained MPD catalytic activity, whereas lysates of vector-transformed controls did not.
Computerized searches of the GenBank and SWISS-PROT databases showed that the C. neoformans deduced peptide sequence was homologous to long-chain alcohol/polyol dehydrogenases from several different micro-organisms. The alignment of C. neoformans MPD with other alcohol dehydrogenases from yeast revealed a conserved region around amino acids 190200 which contains the typical GXGXXG pattern found in the coenzyme-binding domain of dehydrogenases (Wierenga & Hol, 1983 ). The deduced C. neoformans MPD sequence also contained the conserved Cys residues at positions 55 and 136 and the conserved His residue at position 78, which together form the active site for the zinc ligand. The deduced MPD sequence also contained Cys residues at positions 110, 113, 116 and 124, which are similar to the conserved Cys residues in the second zinc-binding site in yeast alcohol dehydrogenase (Jornvall et al., 1987
). Comparison of C. neoformans MPD with other alcohol dehydrogenases also revealed a tetrameric quaternary structure similar to yeast alcohol dehydrogenases, which agrees with the experimental data. Once we noted the structural similarities between C. neoformans MPD and other alcohol dehydrogenases, we tested C. neoformans MPD for alcohol dehydrogenase activity and found that it reduced ethanol at 56% the rate of mannitol 1-phosphate. The ethanol activity was not affected by the addition of salts or EDTA. The Km for ethanol was higher than that for mannitol 1-phosphate, suggesting more efficient binding of mannitol 1-phosphate by the enzyme. Also, horse-liver alcohol dehydrogenase purchased from Sigma reduced NAD in the presence of mannitol 1-phosphate (at 23% the rate of ethanol reduction; data not shown). Based on all the similarities summarized above, we concluded that C. neoformans MPD is a member of the zinc-containing long-chain alcohol dehydrogenase family.
Availability of the C. neoformans MPD cDNA enabled us to begin to examine the functions of MPD. One approach was to examine the phenotypic consequences of overproducing C. neoformans MPD in S. cerevisiae. Chaturvedi et al. (1997) showed that overproduction of bacterial MPD conferred on S. cerevisiae YPH252 gpd1
::leu2 transformants the abilities to convert glucose to mannitol and to grow in high salt concentrations. However, we found that lysates of the S. cerevisiae transformants contained very low levels of MPD catalytic activity. Therefore, the observations that plasmid pYCnmpd did not confer on S. cerevisiae YPH252 the ability to synthesize mannitol from glucose or on S. cerevisiae YPH252 gpd1
::leu2 the ability to grow in hypertonic media may have been due to inefficient heterologous expression rather than implying that MPD does not catalyse a key step in mannitol biosynthesis in C. neoformans.
We also measured MPD mRNA levels in C. neoformans cells that were cultured in glucose or mannitol or that were subjected to temperature and osmotic stresses. If C. neoformans synthesizes and catabolizes mannitol via the unidirectional mannitol cycle originally proposed by Hult et al. (1980) and if MPD is a key component of the biosynthetic arm of this pathway, one would predict that MPD mRNA levels would be higher in glucose-grown C. neoformans cells than in mannitol-grown cells. We found, however, almost as much MPD mRNA in C. neoformans cells cultured in SC-mannitol as in cells cultured in SC-glucose, which was consistent with the observation by Niehaus & Flynn (1994)
that mannitol-grown C. neoformans contained both MPD and M1Pase. We also found that glucose- and mannitol-grown cells of the mannitol-underproducing mutant C. neoformans MLP contained as much MPD mRNA as did wild-type C. neoformans H99. Although this result implies that inability to express MPD mRNA was not responsible for mannitol underproduction in C. neoformans MLP, we have not ruled out a loss of function mutation in the gene encoding MPD in C. neoformans MLP. Lastly, because earlier studies suggested that mannitol may function in C. neoformans as an intracellular stress protectant (Chaturvedi et al., 1996a
), we also examined MPD mRNA levels in C. neoformans cells exposed to temperature and osmotic stresses and we found that MPD mRNA levels increased when C. neoformans was cultured in YPD supplemented with 0·2 or 0·4 M NaCl (but not higher levels) and when the incubation temperature was increased from 25 °C to 30 °C or 37 °C.
Although the results summarized above suggested that MPD may play a role in environmental stress responses, it was not possible to draw definitive conclusions about the functions of MPD in C. neoformans. Therefore, the logical next step will be to generate and analyse C. neoformans mutants lacking MPD. Since targeted gene disruption is now feasible in C. neoformans (Chang et al., 1996 ; Lodge et al., 1994
), we used the MPD cDNA to clone the corresponding structural gene (MPD1) by hybridization. The coding sequence of this gene was identical to that of the MPD cDNA except for the presence of three introns, and a potential TATA box was present 99 bp upstream and an adenosine residue (consistent with the Kozak model of translation initiation in S. cerevisiae (Kozak, 1986
) was present 3 bp upstream of the putative ATG start codon. Since genomic Southern hybridization indicated that MPD1 is a single-copy gene, construction of C. neoformans mpd1 null mutants should be technically feasible and analysis of these mutants should provide definitive information about the functions of MPD in C. neoformans.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72, 248-254.[Medline]
Brown, A. T. & Bowles, R. D. (1977). Polyol metabolism by a caries-conducive Streptococcus: purification and properties of a nicotinamide adenine dinucleotide-dependent mannitol-1-phosphate dehydrogenase. Infect Immun 16, 163-173.[Medline]
Chang, Y. C., Penoyer, L. A. & Kwon-Chung, J. K. (1996). The second capsule gene of Cryptococcus neoformans, CAP64, is essential for virulence. Infect Immun 64, 1977-1983.[Abstract]
Chaturvedi, V., Flynn, T., Niehaus, W. G. & Wong, B. (1996a). Stress tolerance and pathogenic potential of a mannitol mutant of Cryptococcus neoformans. Microbiology 142, 937-943.[Abstract]
Chaturvedi, V. P., Wong, B. & Newman, S. L. (1996b). Oxidative killing of Cryptococcus neoformans by human neutrophils: evidence that fungal mannitol protects by scavenging oxidants. J Immunol 156, 3836-3840.[Abstract]
Chaturvedi, V., Bartiss, A. & Wong, B. (1997). Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol-defective mutant from high-salt and oxidative stress. J Bacteriol 179, 157-162.[Abstract]
Cox, G. M., Rude, T. H., Dykstra, C. C. & Perfect, J. R. (1995). The actin gene from Cryptococcus neoformans: structure and phylogenetic analysis. J Med Vet Mycol 33, 261-266.[Medline]
Eriksson, P., Andre, L., Ansell, R., Bloomberg, A. & Alder, L. (1995). Cloning and characterization of GPD2, a second gene encoding sn-glycerol-3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1. Mol Microbiol 17, 96-107.
Fischer, R., Von Strandmann, R. P. & Hengstenberg, W. (1991). Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the enzymeIIImtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes. J Bacteriol 173, 3709-3715.[Medline]
Foreman, J. E. & Niehaus, W. G. (1985). Zn2+-induced cooperativity of mannitol-1-phosphate dehydrogenase from Aspergillus parasiticus. J Biol Chem 260, 10019-10022.
Gwynne, D. I., Buxton, F. P., Sibley, S., Davies, R. W., Lockington, R., Scazzocchio, A. & Sealy-Lewis, H. M. (1987). Comparison of the cis-acting control regions of two coordinately controlled genes involved in ethanol utilization in Aspergillus nidulans. Gene 51, 205-216.[Medline]
Hult, K., Veide, A. & Gatenbeck, S. (1980). The distribution of the NADPH regenerating mannitol cycle among fungal species. Arch Microbiol 128, 253-255.[Medline]
Jennings, D. H. (1984). Polyol metabolism in fungi. Adv Microb Physiol 25, 149-193.[Medline]
Jornvall, H., Persson, B. & Jeffery, J. (1987). Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem 167, 195-201.[Abstract]
Kiser, R. C. & Niehaus, W. G. (1981). Purification and kinetic characterization of mannitol-1-phosphate dehydrogenase from Aspergillus niger. Arch Biochem Biophys 211, 613-621.[Medline]
Kozak, M. (1986). Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Lodge, J. K., Jackson-Machelski, E., Toffaletti, D. L., Perfect, J. R. & Gordon, J. I. (1994). Targeted gene replacement demonstrates that myristoyl-CoA:protein N-myristoyltransferase is essential for viability of Cryptococcus neoformans. Proc Natl Acad Sci U S A 91, 12008-12012.
McKnight, G. L., Kato, H., Upshall, A., Parker, M. D., Saari, G. & OHara, P. J. (1985). Identification and molecular analysis of a third Aspergillus nidulans alcohol dehydrogenase gene. EMBO J 4, 2093-2099.[Abstract]
Niehaus, W. G. & Flynn, T. (1994). Regulation of mannitol biosynthesis and degradation by Cryptococcus neoformans. J Bacteriol 176, 651-655.[Abstract]
Novotny, M. J., Reizer, J., Esch, F. & Saier, M. H.Jr (1984). Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli. J Bacteriol 159, 986-990.[Medline]
OFarrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007-4021.[Abstract]
Parker, R. & Patterson, B. (1987). Molecular Biology of RNA: New Perspectives. Edited by M. Inouye & B. S. Dudock. New York: Academic Press.
Saliola, M., Gonnella, R., Mazzony, C. & Falcone, C. (1991). Two genes encoding putative mitochondrial alcohol dehydrogenases are present in the yeast Kluyveromyces lactis. Yeast 7, 391-400.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schmatz, D. M. (1997). The mannitol cycle in Eimeria. Parasitology 114S, S81-S89.[Medline]
Shen, H. D., Choo, K. B., Lee, H. H., Hsieh, J. C., Lee, W. R. & Han, S. H. (1991). The 40-kilodalton allergen of Candida albicans is an alcohol dehydrogenase: molecular cloning and immunological analysis using monoclonal antibodies. Clin Exp Allergy 21, 675-681.[Medline]
Sherman, F., Fink, G. R. & Hicks, J. B. (1986). Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Tarczynski, M. C., Jensen, R. G. & Bohnert, H. J. (1993). Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science 259, 506-510.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[Abstract]
Wang, H. T., Rahaim, P., Robbins, P. & Yocum, R. R. (1994). Cloning, sequence, and disruption of the Saccharomyces diastaticus DAR1 gene encoding a glycerol-3-phosphate dehydrogenase. J Bacteriol 176, 7091-7095.[Abstract]
Wang, S. Y. & Le Tourneau, D. (1972). Mannitol biosynthesis in Sclerotinia sclerotiorum. Arch Mikrobiol 81, 91-99.[Medline]
Wierenga, R. K. & Hol, W. G. J. (1983). Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant. Nature 302, 842-844.[Medline]
Woloshuk, C. P. & Payne, G. A. (1994). The alcohol dehydrogenase gene adh1 is induced in Aspergillus flavus grown on medium conducive to aflatoxin biosynthesis. Appl Environ Microbiol 60, 670-676.[Abstract]
Wong, B., Brauer, K. L., Tsai, R. R. & Jayasimhulu, K. (1989). Increased amounts of the Aspergillus metabolite D-mannitol in tissues and serum of animals with experimental aspergillosis. J Infect Dis 160, 95-103.[Medline]
Wong, B., Perfect, J. R., Beggs, S. & Wright, K. A. (1990). Production of the hexitol D-mannitol by Cryptococcus neoformans in vitro and in rabbits with experimental meningitis. Infect Immun 58, 1664-1670.[Medline]
Received 11 February 2000;
revised 16 June 2000;
accepted 6 July 2000.