An STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant

Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species identified in its genus. At 25 degrees P. marneffei exhibits true filamentous growth, while at 37 degrees P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. Members of the STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number of fungi. We have cloned a homolog of the S. cerevisiae STE12 gene from P. marneffei, stlA, which is highly conserved. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12alpha genes, form a distinct subclass of STE12 homologs that have a C2H2 zinc-finger motif in addition to the homeobox domain that defines STE12 genes. To examine the function of stlA in P. marneffei, we isolated a number of mutants in the P. marneffei-type strain and, in combination with selectable markers, developed a highly efficient DNA-mediated transformation procedure and gene deletion strategy. Deletion of the stlA gene had no detectable effect on vegetative growth, asexual development, or dimorphic switching in P. marneffei. Despite the lack of a detectable function, the P. marneffei stlA gene complemented the sexual defect of an A. nidulans steA mutant. In addition, substitution rate estimates indicate that there is a significant bias against nonsynonymous substitutions. These data suggest that P. marneffei may have a previously unidentified cryptic sexual cycle.     

A basic helix-loop-helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei

Members of the APSES protein group are basic helix-loop-helix (bHLH) proteins that regulate processes such as mating, asexual sporulation and dimorphic growth in fungi. Penicillium marneffei is a human pathogen and is the only member of its genus to display a dimorphic growth transition. At 25 degrees C, P. marneffei grows with a filamentous morphology and produces asexual spores from multicellular con-idiophores. At 37 degrees C, the filamentous morphology is replaced by yeast cells that reproduce by fission. We have cloned and characterized an APSES protein-encoding gene from P. marneffei that has a high degree of similarity to Aspergillus nidulans stuA. Deletion of stuA in P. marneffei showed that it is required for metula and phialide formation during conidiation but is not required for dimorphic growth. This suggests that APSES proteins may control processes that require budding (formation of the metulae and phialides, pseudohyphal growth in Saccharomyces cerevisiae and dimorphic growth in Candida albicans) but not those that require fission (dimorphic growth in P. marneffei). The A. nidulans DeltastuA mutant has defects in both conidiation and mating. The P. marneffei stuA gene was capable of complementing the conidiation defect but could only inefficiently complement the sexual defects of the A. nidulans mutant. This suggests that the P. marneffei gene, which comes from an asexual species, has diverged significantly from the A. nidulans gene with respect to sexual but not asexual development.     

AreA controls nitrogen source utilisation during both growth programs of the dimorphic fungus Penicillium marneffei

The opportunistic pathogen Penicillium marneffei displays a temperature-dependent dimorphic switching program with saprophytic hyphal growth at 25 °C and yeast growth at 37 °C. The areA gene of P. marneffei has been isolated and found to be required for the utilisation of nonpreferred nitrogen sources during both growth programs of P. marneffei, albeit to differing degrees. Based on this functional characterisation and high degree of sequence conservation with other fungal GATA factors, P. marneffei areA represents an orthologue of Aspergillus nidulans areA and Neurospora crassa NIT2. Based on this study it is proposed that AreA is likely to contribute to the pathogenicity of P. marneffei by facilitating growth in the host environment and regulating the expression of potential virulence factors such as extracellular proteases.     

Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans.

Genetic analysis of 119 acetate non-utilizing (acu) mutants in Aspergillus nidulans revealed ten new loci affecting acetate metabolism in addition to the three previously recognized on the basis of resistance to fluoroacetate and acetate non-utilization. The enzyme lesions associated with mutations at seven of the acu loci are described. These are: facA (= acuA), acetyl-CoA synthase; acuD, isocitrate lyase; acuE, malate synthase; acuF, phosphoenolpyruvate carboxykinase; acuG, fructose 1,6-diphosphatase; acuK and acuM, malic enzyme. The acu loci have been mapped and are widely distributed over the genome of A. nidulans. Close linkage has only been found between acuA and acuD (less than 1% recombination). There is no evidence for any pleiotropic mutation in that region affecting the expression of both these genes. Poor induction of the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase in mutants lacking acetyl-CoA synthase, and also in the other two classes of fluoroacetate-resistant mutants, indicates that the inducer, acetate, may be metabolized to a true metabolic inducer, perhaps acetyl-CoA, to effect formation of the enzymes. There is no evidence of any other class of pleiotropic recessive acu mutations affecting the expression of the acuD and acuE genes, which are therefore thought to be subject to negative rather than positive control.     

The G-protein alpha-subunit GasC plays a major role in germination in the dimorphic fungus Penicillium marneffei

The opportunistic human pathogen Penicillium marneffei exhibits a temperature-dependent dimorphic switch. At 25 degrees, multinucleate, septate hyphae that can undergo differentiation to produce asexual spores (conidia) are produced. At 37 degrees hyphae undergo arthroconidiation to produce uninucleate yeast cells that divide by fission. This work describes the cloning of the P. marneffei gasC gene encoding a G-protein alpha-subunit that shows high homology to members of the class III fungal Galpha-subunits. Characterization of a DeltagasC mutant and strains carrying a dominant-activating gasC(G45R) or a dominant-interfering gasC(G207R) allele show that GasC is a crucial regulator of germination. A DeltagasC mutant is severely delayed in germination, whereas strains carrying a dominant-activating gasC(G45R) allele show a significantly accelerated germination rate. Additionally, GasC signaling positively affects the production of the red pigment by P. marneffei at 25 degrees and negatively affects the onset of conidiation and the conidial yield, showing that GasC function overlaps with functions of the previously described Galpha-subunit GasA. In contrast to the S. cerevisiae ortholog Gpa2, our data indicate that GasC is not involved in carbon or nitrogen source sensing and plays no major role in either hyphal or yeast growth or in the switch between these two forms.     

The CDC42 homolog of the dimorphic fungus Penicillium marneffei is required for correct cell polarization during growth but not development

The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved CDC42 homolog from P. marneffei named cflA. By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct cflA function is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression of cflA alleles in Aspergillus nidulans prevented conidiation.     

The regulation of peroxisomal enzyme systems of Tetrahymena pyriformis by fatty acid composition, glucose and oxygen in the medium

The effect of the chain length of fatty acids on peroxisomal enzyme activities of Tetrahymena pyriformis was investigated. The growth of cells and the activities of peroxisomal enzymes were inhibited markedly by the addition of medium-chain fatty acids (C6-C12) to the culture medium, whereas the addition of longer-chain fatty acids (C14-C18) resulted in a slight increase of growth and in the marked stimulation of enzyme activities concerned with fatty acid beta-oxidation and the glyoxylate cycle in peroxisomes. Peroxisomal beta-oxidation (fatty acyl-CoA oxidase) was more potent towards longer-chain fatty acids than the mitochondrial activity (fatty acyl-CoA dehydrogenase). The induction of the peroxisomal beta-oxidation system by palmitate was repressed both by the addition of glucose and the aeration of the culture medium, whereas that of the peroxisomal glyoxylate cycle was repressed only by the addition of glucose to the medium. These results indicate that peroxisomal enzyme systems related to the beta-oxidation of fatty acids and the glyoxylate cycle are regulated by the compositions of fatty acids, glucose, and oxygen in the medium.     

Expression of a functionally active cardiac fatty acid-binding protein in the yeast,Saccharomyces cerevisiae

Summary The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size to native protein as judged from SDS-polyacrylamide gel electrophoresis, was reached after approximately 16 hours of induction. Analysis of particulate and soluble subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind ¹⁴C-oleic acid in an in vitro assay. Growth of the transformants on galactose as the carbon source was significantly retarded at 37°C. Whereas the fatty acid pattern of total lipids was not altered in transformed cells, desaturation of exogenously added ¹⁴C-palmitic acid was significantly reduced both at 30 and 37°C. The lowest percentage of radioactively labeled unsaturated fatty acids was found in the phospholipid fraction.     

Insights into the pathogenicity of Penicillium marneffei.

Penicillium marneffei is a significant pathogen of AIDS patients in Southeast Asia. This fungus is unique in that it is the only dimorphic member of the genus. Pathogenesis of P. marneffei requires the saprobic mold form to undergo a morphological change upon tissue invasion. The in vivo form of this fungus reproduces as a fission yeast that capably evades the host immune system. The processes that control these morphological changes, better termed as phase transition, can be replicated in vitro by incubation of the mold form at 37 degrees C. The unidentified molecular mechanisms regulating phase transition in this fungus are now being uncovered using modern methodologies and novel strategies. A better comprehension of these underlying regulatory pathways will provide insight into eukaryotic cellular development as well as the potential factors responsible for infections caused by P. marneffei and other fungi. Such knowledge may lead to better chemotherapeutic interventions of fungal diseases.     

Biochemical and structural studies of malate synthase from Mycobacterium tuberculosis

Establishment or maintenance of a persistent infection by Mycobacterium tuberculosis requires the glyoxylate pathway. This is a bypass of the tricarboxylic acid cycle in which isocitrate lyase and malate synthase (GlcB) catalyze the net incorporation of carbon during growth of microorganisms on acetate or fatty acids as the primary carbon source. The glcB gene from M. tuberculosis, which encodes malate synthase, was cloned, and GlcB was expressed in Escherichia coli. The influence of media conditions on expression in M. tuberculosis indicated that this enzyme is regulated differentially to isocitrate lyase. Purified GlcB had K(m) values of 57 and 30 microm for its substrates glyoxylate and acetyl coenzyme A, respectively, and was inhibited by bromopyruvate, oxalate, and phosphoenolpyruvate. The GlcB structure was solved to 2.1-A resolution in the presence of glyoxylate and magnesium. We also report the structure of GlcB in complex with the products of the reaction, coenzyme A and malate, solved to 2.7-A resolution. Coenzyme A binds in a bent conformation, and the details of its interactions are described, together with implications on the enzyme mechanism.     

Evaluation of the tetracycline promoter system for regulated gene expression in Kluyveromyces marxianus

A tetracycline repressible promoter system designed for Saccharomyces cerevisiae was evaluated for use in Kluyveromyces marxianus. A plasmid was constructed containing the Escherichia coli beta-glucuronidase (gus) gene cloned downstream of the yeast tet-off promoter, the tetR-VP16 activator protein gene, and the URA3 gene for selection. The tet-off promoter-gus construct was integrated into the chromosomal DNA and tested under varying growth conditions in complex medium. The repressors tetracycline and doxycycline were both found to be effective for inhibiting gene expression. Doxycycline levels of 0.5 microg/mL or greater were sufficient to nearly completely suppress Gus synthesis. For most transformants, the induction ratio was approximately 2,000-fold. The tet-off promoter was effective at 30, 37, and 42 degrees C, although the overall Gus activity was highest at 37 degrees C. During exponential growth, little product was formed; expression increased dramatically in late exponential and early stationary phase. The promoter thus shows promise for protein synthesis following cell growth. No inducer is required and the repressor is only needed to prevent expression during the seed culture.     

Regulation of the acuF Gene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous Fungus Aspergillus nidulans

Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle. Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability of A. nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S. cerevisiae. The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S. cerevisiae Hap complex and the mammalian NFY complex.