HWP1 functions in the morphological development of Candida albicans downstream of EFG1, TUP1, and RBF1.

The morphological plasticity of Candida albicans is an important determinant of pathogenicity, and nonfilamentous mutants are avirulent. HWP1, a hypha-specific gene, was identified in a genetic screen for developmentally regulated genes and encodes a cell surface protein of unknown function. Heterozygous and homozygous deletions of HWP1 resulted in a medium-conditional defect in hyphal development. HWP1 expression was blocked in a Deltaefg1 mutant, reduced in an Deltarbf1 mutant, and derepressed in a Deltatup1 mutant. Therefore, HWP1 functions downstream of the developmental regulators EFG1, TUP1, and RBF1. Mutation of CPH1 had no effect on HWP1 expression, suggesting that the positive regulators of hyphal development, CPH1 and EFG1, are components of separate pathways with different target genes. The expression of a second developmentally regulated gene, ECE1, was similarly regulated by EFG1. Since ECE1 is not required for hyphal development, the regulatory role of EFG1 apparently extends beyond the control of cell shape determinants. However, expression of ECE1 was not influenced by TUP1, suggesting that there may be some specificity in the regulation of morphogenic elements during hyphal development.

PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis.

Candida albicans, like many fungi, exhibits morphological plasticity, a property which may be related to its biological capacity as an opportunistic pathogen of humans. Morphogenesis and alterations in cell shape require integration of many cellular functions and occur in response to environmental signals, most notably pH and temperature in the case of C. albicans. In the course of our studies of differential gene expression associated with dimorphism of C. albicans, we have isolated a gene, designated PHR1, which is regulated in response to the pH of the culture medium. PHR1 expression was repressed at pH values below 5.5 and induced at more alkaline pH. The predicted amino acid sequence of the PHR1 protein was 56% identical to that of the Saccharomyces cerevisiae Ggp1/Gas1 protein, a highly glycosylated cell surface protein attached to the membrane via glycosylphosphatidylinositol. A homozygous null mutant of PHR1 was constructed and found to exhibit a pH-conditional morphological defect. At alkaline pH, the mutant, unlike the parental type, was unable to conduct apical growth of either yeast or hyphal growth forms. This morphological aberration was not associated with defective cytoskeletal polarization or secretion. The results suggest that PHR1 defines a novel function required for apical cell growth and morphogenesis.

Use of URA3 as a reporter of gene expression in C. albicans.

The C. albicans URA3 gene was tested as a reporter of gene expression. An integrating vector was constructed which contained ADE2 as a selectable marker together with a truncated form of URA3 lacking the first three codons. A DNA fragment containing the promoter and the first 90 codons of the C. albicans CEF3 gene was inserted into the unique XhoI site 5' to URA3 in order to provide an in-frame translational fusion. The functionality of the fusion gene was tested following integration of a single copy of the plasmid into the ADE2 locus. The fusion gene was shown to complement a ura3 deletion mutation and to produce orotidine 5'-monophosphate decarboxylase activity (OMP), which is encoded by URA3. Expression of the fusion gene was appropriately regulated by the growth rate and utilized the same transcriptional start sites as the native CEF3 gene. The results demonstrated that URA3 provides a sensitive and versatile reporter gene for use in C. albicans.

Cloning and characterization of ECE1, a gene expressed in association with cell elongation of the dimorphic pathogen Candida albicans.

The gene ECE1 (extent of cell elongation 1) was isolated by differential hybridization screening of a Candida albicans cDNA library by using probes derived from populations of yeast cells or hyphae. Expression of this gene was not detected when C. albicans grew as a budding yeast cell but was observed within 30 min after cells had been induced to form hyphae. In all strains tested, regardless of the induction signal, ECE1 expression correlated with the extent of cell elongation. The genomic version of ECE1 was cloned and sequenced. The deduced 271-amino-acid polypeptide consisted of eight tandem repeats of a degenerate 34-amino-acid sequence which contained no discernible homology with other known sequences. An ECE1 null mutant displayed no morphological alterations, which demonstrated that ECE1 is not essential for cell elongation or hypha formation despite the strict morphological association of its expression.

Fungus-specific translation elongation factor 3 gene present in Pneumocystis carinii.

Historically, Pneumocystis carinii pneumonia has been the most frequent cause of morbidity and mortality in patients with AIDS. Antiprotozoan drugs are effective in the treatment and prophylaxis of P. carinii pneumonia, which lends credence to the widely held view that P. carinii is a protozoan. However, recent genetic evidence suggests that P. carinii should be classified as a fungus. Translation elongation factor 3 (EF-3) is an essential, soluble translation component which is unique to fungal protein synthesis and is not required for protein synthesis in other eukaryotes. We have identified and isolated a gene for EF-3 from P. carinii, adding more evidence for this organism's assignment as a fungus.

Isolation and sequence analysis of the gene for translation elongation factor 3 from Candida albicans.

Elongation factor 3 (EF-3) is a unique and essential component of the translational system in fungi. The gene, CEF-3, encoding elongation factor 3 has been isolated from the dimorphic fungus Candida albicans. A heterologous gene probe containing the coding region of the EF-3 gene from Saccharomyces cerevisiae (YEF-3) was used to screen three Candida albicans genomic DNA libraries. The nucleotide sequences of four partial clones were determined and combined for a full-length of 3,671 base pairs (bp). A continuous open reading frame (ORF) of 3,147 bp encoding a predicted protein of 1,049 amino acids and Mr of 116,739 daltons has been identified. A transcript of 3,400 nucleotides is seen in Northern blot hybridization of Candida albicans total RNA using a CEF-3 gene probe. The single locus CEF-3 gene maps to chromosome 5 in the genome. Comparison of the deduced amino acid sequences of CEF-3 and YEF-3 shows 77.6%. identity. A higher degree of identity, 86.5%, is found when comparing the carboxy-terminal portions of the two proteins. At the nucleotide level, comparison of the coding regions of the two genes exhibit 79% identity while the upstream and downstream regions show 46% and 40% identity, respectively.

The isolation and characterization of a calmodulin-encoding gene (CMD1) from the dimorphic fungus Candida albicans.

Candida albicans is a dimorphic, opportunistic pathogen of humans, and calcium and calmodulin have been implicated in its morphogenic transition. The C. albicans calmodulin-encoding gene, CMD1, was isolated from cDNA and genomic phage lambda libraries using the Saccharomyces cerevisiae CMD gene as a hybridization probe. Southern-blot hybridization analysis of genomic DNA suggests the existence of only one type of calmodulin gene in C. albicans. Comparison of cDNA and genomic sequences identified a 222-bp intron located immediately after the Met start codon. The predicted amino acid sequence was 60% identical with yeast CMD and 70% identical with CMDs of filamentous fungi and vertebrates. We have localized the CMD1 gene to chromosome 3 using the contour-clamped homogeneous electric field gel electrophoresis. The CMD1 gene hybridized to a single 650-nucleotide transcript which was present in equivalent amounts in both the yeast and hyphal forms of the organism.

Both genes for EF-1 alpha in Candida albicans are translated.

In previous work, we showed that Candida albicans has two genes, TEF-1 and TEF-2, which encode identical polypeptides for the highly conserved, essential, protein synthesis factor EF-1 alpha (Breviario et al., 1988). This result prompted questions as to whether C. albicans preferentially uses one of the genes over the other and whether both genes are actually translated into protein. Gene-specific sequence differences in the untranslated portion of each gene made it possible to prepare gene-specific oligonucleotide hybridization probes. Results with the probes showed that the relative steady-state mRNA levels of the two genes were equivalent and that the mRNA for each gene was present in active translation complexes.

Sequence analysis and expression of the two genes for elongation factor 1 alpha from the dimorphic yeast Candida albicans.

Two Candida albicans genes that encode the protein synthesis factor elongation factor 1 alpha (EF-1 alpha) were cloned by using a heterologous TEF1 probe from Mucor racemosus to screen libraries of C. albicans genomic DNA. Sequence analysis of the two clones showed that regions of DNA flanking the coding regions of the two genes were not homologous, verifying the presence of two genes, called TEF1 and TEF2, for EF-1 alpha in C. albicans. The coding regions of TEF1 and TEF2 differed by only five nucleotides and encoded identical EF-1 alpha proteins of 458 amino acids. Both genes were transcribed into mRNA in vivo, as shown by hybridization of oligonucleotide probes, which bound specifically to the 3' nontranslated regions of TEF1 and TEF2, respectively, to C. albicans total RNA in Northern (RNA) blot analysis. The predicted EF-1 alpha protein of C. albicans was more similar to Saccharomyces cerevisiae EF-1 alpha than to M. racemosus EF-1 alpha. Furthermore, codon bias and the promoter and termination signals of the C. albicans EF-1 alpha proteins were remarkably similar to those of S. cerevisiae EF-1 alpha. Taken together, these results suggest that C. albicans is more closely related to the ascomycete S. cerevisiae than to the zygomycete M. racemosus.

Role of lysine methylation in the activities of elongation factor 1 alpha.

Previous work in our laboratory has demonstrated that 19% of the lysine residues in the protein synthesis elongation factor (EF-1 alpha) are methylated when the factor is purified from the mycelial form of the fungus Mucor racemosus. However, the same factor, when purified from spores of M. racemosus, is largely unmethylated. Despite its wide-spread occurrence in a great number of basic proteins, the functional significance of lysine N-methylation remains poorly understood. Spore and mycelial forms of EF-1 alpha were therefore compared in a series of assays to determine their relative affinities for various substrates and cofactors known to interact with the factor during the elongation cycle. The results suggested that hypomethylated and fully methylated EF-1 alpha had equal affinities for GTP, aminoacyl-tRNA, and ribosomes. Also, methylation did not appear to affect the accuracy of translation in an in vitro system. However, experiments did suggest that methylation may affect the ability of the factor to form complexes with other subunits (EF-1 beta gamma) which are known to enhance the overall rate of protein synthesis.

Structure of a ribosomal protein gene in Mucor racemosus.

As an extension of our analysis of the translational apparatus of Mucor racemosus we have isolated a gene encoding a ribosomal protein of Mucor. Based on a method developed for S. cerevisiae, we identified by hybrid selection and in vitro translation a lambda-Charon 4A clone containing the genomic copy of a Mucor ribosomal protein. The gene consisted of two exons of 57 and 387 nucleotides. The two exons were separated by an 131 nucleotide intron. The processed transcript was 714 nucleotides in length and contained a 25 nucleotide untranscribed leader and an 114 nucleotide untranscribed 3'-end. The protein predicted from the nucleotide sequence contained 148 amino acids and exhibited 61% identity with the S19 ribosomal protein of Xenopus laevis. The promoter region of the gene contained sequences highly homologous to the RPG and Homol1 promoter elements found in S. cerevisiae. Southern blot analysis indicated that the Mucor genome contains three copies of this gene.

Cycloheximide efflux in antibiotic-adapted cells of the fungus Mucor racemosus.

Mucor racemosus cells adapted to either cycloheximide or trichodermin were approximately 40-fold more resistant to cycloheximide than nonadapted cells. Ribosomes isolated from adapted and nonadapted cells were equally sensitive to cycloheximide in an in vitro poly(U) translation assay. There was no detectable modification of cycloheximide by adapted cells. Uptake of drug by nonadapted and adapted cells was characterized by a rapid initial accumulation during the first 2 min of incubation with [3H]cycloheximide, followed by a steady-state intracellular drug concentration well below that of the medium. The steady-state drug concentration was approximately 10-fold lower in adapted cells than in nonadapted cells. Treatment of cells with sodium azide or dinitrophenol abolished the difference between uptake of drug by nonadapted and adapted cells and resulted in intracellular drug levels equal to that of the medium. Direct efflux measurements showed that adapted cells loaded with cycloheximide were able to excrete the drug far more rapidly than nonadapted cells. These results suggest that both nonadapted and adapted cells possess an energy-dependent efflux mechanism for transporting cycloheximide and that resistance in adapted cells is due to increased efficiency of transport.

Sequence analysis of the EF-1 alpha gene family of Mucor racemosus.

Our previous studies have shown that Mucor racemosus possesses three genes (TEF-1, -2 and -3) for EF-1 alpha, and that all three genes are transcribed. However, the level of transcription varies markedly between the three genes, with TEF-1 mRNA levels being approximately two fold higher than TEF-3 and 6 fold higher than TEF-2. We have now completed the DNA sequence of both strands of all three genes and have found that these genes are highly homologous. TEF-2 and TEF-3 are more similar to each other than they are to TEF-1. The TEF-2 and the TEF-3 coding regions differ from TEF-1 at 30 and 37 positions respectively out of 1374 nucleotides. Twenty-six of these nucleotide substitutions were common to both TEF-2 and TEF-3, and the majority of the substitutions were clustered in the 5' region of the coding sequences. While the majority of these changes were silent, TEF-2 and TEF-3 differed from TEF-1 by having a lysine instead of a glutamate at amino acid position 41. In addition, TEF-2 and -3, but not TEF-1, each have an intron located near the 5' end of the coding region, although its size and sequence is not conserved between the two genes. All three genes have a conserved intron near the 3' end of the coding region. The sequence data have been analyzed with respect to the structure and function of EF-1 alpha in protein biosynthesis.

The gene and the primary structure of ornithine decarboxylase from Saccharomyces cerevisiae.

The nucleotide sequence was determined for a 3-kilobase genomic fragment containing the ornithine decarboxylase gene of Saccharomyces cerevisiae. The fragment contained two open reading frames. Gene disruption localized the ornithine decarboxylase gene to a 1398-nucleotide open reading frame. Transcription of the yeast gene initiated at several sites 171 to 211 nucleotides 5' of the translational start site. The 3' end of the transcript extended approximately 300 nucleotides beyond the end of the ornithine decarboxylase coding region and contained two copies of the yeast ARS core sequence. Translation of the ornithine decarboxylase gene appeared to initiate at the first AUG condon of the open reading frame based upon translational fusions with the Escherichia coli beta-galactosidase gene. Since no introns were apparent, the 1398-nucleotide open reading frame was predicted to encode a 466-amino acid protein with a calculated Mr = 52,369. The deduced protein differed significantly in size from previous reports on yeast ornithine decarboxylase, but was very similar in size to mammalian ornithine decarboxylase. When the predicted amino acid sequence of yeast ornithine decarboxylase was compared with that of the mouse enzyme, alignment of the sequences revealed that 40% of the amino acid residues were identical. Chou-Fasman predictions of the secondary structure of the two enzymes indicated that secondary structure was also highly conserved.

Expression of three genes for elongation factor 1 alpha during morphogenesis of Mucor racemosus.

Three genes, TEF-1, -2, and -3, encode elongation factor 1 alpha in Mucor racemosus. Neutral and alkaline S1 nuclease analyses revealed that the genetic organization is unique for each of the genes. The number and size of the intervening sequences vary in these closely related genes, which suggests that complex genetic rearrangements gave rise to the elongation factor 1 alpha gene family. Nucleotide sequence data from restriction fragments isolated from the 5' and 3' ends of TEF-2 and -3 confirmed the presence of a second intervening sequence in these genes. These data along with S1 nuclease mapping revealed a region at the 3' end of the three genes which was predicted to be transcribed but untranslated. Unique oligonucleotides containing 19 bases were synthesized to hybridize to this unique trailer region in the elongation factor 1 alpha transcripts. These oligonucleotides were used as probes in standard Northern analysis of RNA purified from M. racemosus cells of several morphological types. It was determined that all three genes were expressed in the cell morphological types studied. However, the accumulated level of transcript derived from each gene varied considerably, with TEF-1 mRNA present in approximately twofold greater quantity than the TEF-3 transcript and up to sixfold greater quantity than TEF-2. The level of TEF-1 and -2 mRNA varied little among the cell morphological types studied, whereas TEF-3 mRNA was present in twofold greater quantity in sporangiospores than in either germlings or yeast cells which had been induced to undergo morphogenesis to hyphae. These data suggest that there is differential expression of the genes encoding elongation factor 1 alpha in M. racemosus. At least one gene, TEF-3, shows a morphology-specific pattern of transcript accumulation.

The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus.

We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus. The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons. The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping. The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina. These proteins shared nearly 85% homology. A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms. Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M. racemosus. Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA.

Identification of epsilon-N-mono-, epsilon-N-di-, and epsilon-N-trimethyllysine by high-performance liquid chromatography.

Dansyl derivatives of epsilon-N-mono-, epsilon-N-di-, and epsilon-N-trimethyllysine were resolved from other amino acids in proteins by the use of high-performance liquid chromatography. The system was tested with amino acid standard combinations as well as with acid-hydrolyzed proteins known to contain methylated residues. In all cases the methylated lysines were well resolved.

Trichodermin esterase activity and trichodermin resistance in Mucor racemosus.

Mucor racemosus exhibited inducible phenotypic resistance toward the protein synthesis inhibitor trichodermin. Induction of resistance was elicited by exposure to trichodermin or to cycloheximide. Both adapted and nonadapted cells took up [14C]trichodermin from the medium. Trichodermin was found to be rapidly deacetylated to trichodermol upon entering the cell. Adapted cells deacetylated the drug more rapidly than nonadapted cells both in vivo and in vitro. The trichodermol resulting from deacetylation appeared in the medium, but the growth of adapting cells began well before the total conversion of trichodermin to trichodermol. Based on these data and the observation that trichodermol was a poor inhibitor of Mucor, adaptation appears to result from deacylation of the active antibiotic.

Three genes for the elongation factor EF-1 alpha in Mucor racemosus.

We cloned three genes from Mucor racemosus coding for protein synthesis elongation factor 1 alpha (EF-1 alpha). A 110-base-pair (bp) EF-1 alpha-specific cDNA clone was identified by hybrid-selected translation. The nucleotide sequence of the cDNA showed significant homology to a region of the Saccharomyces cerevisiae genes for EF-1 alpha (TEF1 and TEF2). The cDNA was used to isolate an 850-bp EcoRI genomic DNA fragment containing a portion of the EF-1 alpha gene. Screening of a lambda/M. racemosus genomic DNA bank with the 850-bp EcoRI probe resulted in the identification of three DNA fragments containing a common 850-bp EcoRI fragment within a short overlapping region. S1 nuclease analysis of the three EF-1 alpha DNA fragments showed that the EF-1 alpha transcript covered the short overlapping region in the clones. Restriction fragments purified from flanking regions in each clone were used to probe a HindIII digest of M. racemosus genomic DNA. Each flanking probe hybridized to one of three DNA fragments which hybridized to the 850-bp EF-1 alpha-specific probe. Nucleotide sequence data from two random "shotgun clones" of one of the three genes show good homology to two regions of S. cerevisiae TEF1. The data indicate the presence of three genes for EF-1 alpha in M. racemosus located at unique sites in the genome.

Inducible phenotypic multidrug resistance in the fungus Mucor racemosus.

The dimorphic fungus Mucor racemosus exhibited a single-step, inducible resistance to cycloheximide, trichodermin, and amphotericin B. Cells adapted to inhibitory levels of the antibiotics after 12 to 40 h. The adaptation involved all the cells in the population and was not the result of the selection of resistant mutants. Adaptation to one drug provided cross resistance to other, dissimilar drugs. Resistance was lost within several generations of growth in the absence of the inhibitors.