Candida albicans White-Opaque Switching Influences Virulence but Not Mating during Oropharyngeal Candidiasis.

The capacity of Candida albicans to switch reversibly between the white phenotype and the opaque phenotype is required for the fungus to mate. It also influences virulence during hematogenously disseminated candidiasis. We investigated the roles of the mating type loci (MTL) and white-opaque switching in the capacity of C. albicans to mate in the oropharynx and cause oropharyngeal candidiasis (OPC). When immunosuppressed mice were orally infected with mating-competent opaque a/a and α/α cells either alone or mixed with white cells, no detectable mating occurred, indicating that the mating frequency was less than 1.6 × 10-6 Opaque cells were also highly attenuated in virulence; they either were cleared from the oropharynx or switched to the white phenotype during OPC. Although there were strain-to-strain differences in the virulence of white cells, they were consistently more virulent than opaque cells. In vitro studies indicated that relative to white cells, opaque cells had decreased capacity to invade and damage oral epithelial cells. The reduced invasion of at least one opaque strain was due to reduced surface expression of the Als3 invasin and inability to activate the epidermal growth factor receptor, which is required to stimulate the epithelial cell endocytic machinery. These results suggest that mating is a rare event during OPC because opaque cells have reduced capacity to invade and damage the epithelial cells of the oral mucosa.

Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms.

In the screening of natural plant extracts for antifungal activity, assessment of their effects on the growth of cells in suspension or in the wells of microtiter plates is expedient. However, microorganisms, including Candida albicans, grow in nature as biofilms, which are organized cellular communities with a complex architecture capable of conditioning their microenvironment, communicating, and excluding low- and high-molecular-weight molecules and white blood cells. Here, a confocal laser scanning microscopy (CLSM) protocol for testing the effects of large numbers of agents on biofilm development is described. The protocol assessed nine parameters from a single z-stack series of CLSM scans for each individual biofilm analyzed. The parameters included adhesion, thickness, formation of a basal yeast cell polylayer, hypha formation, the vertical orientation of hyphae, the hyphal bend point, pseudohypha formation, calcofluor white staining of the extracellular matrix (ECM), and human white blood cell impenetrability. The protocol was applied first to five plant extracts and derivative compounds and then to a collection of 88 previously untested plant extracts. They were found to cause a variety of phenotypic profiles, as was the case for 64 of the 88 extracts (73%). Half of the 46 extracts that did not affect biofilm thickness affected other biofilm parameters. Correlations between specific effects were revealed. The protocol will be useful not only in the screening of chemical libraries but also in the analysis of compounds with known effects and mutations.

Interferon regulatory factor 6 regulates keratinocyte migration.

Interferon regulatory factor 6 (Irf6) regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post-conception failed to close their wound compared with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell-cell and cell-matrix adhesions were investigated. We show that wild-type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPase-activating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration.

Comparison of Switching and Biofilm Formation between MTL-Homozygous Strains of Candida albicans and Candida dubliniensis.

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO2 induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO2, exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved.

Role of Tec1 in the development, architecture, and integrity of sexual biofilms of Candida albicans.

MTL-homozygous ( A: / A: or α/α) white cells form a complex sexual biofilm that exhibits the same architecture as that of MTL-heterozygous ( A: /α) pathogenic biofilms. However, the former is regulated by the mitogen-activated protein (MAP) kinase pathway, while the latter is regulated by the Ras1/cyclic AMP (cAMP) pathway. We previously demonstrated that in the formation of an MTL-homozygous, mature (48 h) sexual biofilm in RPMI 1640 medium, the MAP kinase pathway targets Tec1 rather than Cph1, the latter of which is the target of the same pathway, but for the opaque cell mating response. Here we continued our analysis of the role of Tec1 by comparing the effects of deleting TEC1 on initial adhesion to silicone elastomer, high-resolution confocal microscopy assessments of the stages and cellular phenotypes during the 48 h of biofilm development, human white cell penetration, and biofilm fragility. We show that although Tec1 plays only a minor role in initial adhesion to the silicone elastomer, it does play a major role in the growth of the basal yeast cell polylayer, vertical extension of hyphae and matrix deposition in the upper portion of the biofilm, final biofilm thickness, penetrability of human white blood cells, and final biofilm integrity (i.e., resistance to fluid flow). These results provide a more detailed description of normal biofilm development and architecture and confirm the central role played by the transcription factor Tec1 in the biofilm model employed here.

Huntingtin regulates Ca(2+) chemotaxis and K(+)-facilitated cAMP chemotaxis, in conjunction with the monovalent cation/H(+) exchanger Nhe1, in a model developmental system: insights into its possible role in Huntington׳s disease.

Huntington׳s disease is a neurodegenerative disorder, attributable to an expanded trinucleotide repeat in the coding region of the human HTT gene, which encodes the protein huntingtin. These mutations lead to huntingtin fragment inclusions in the striatum of the brain. However, the exact function of normal huntingtin and the defect causing the disease remain obscure. Because there are indications that huntingtin plays a role in Ca(2+) homeostasis, we studied the deletion mutant of the HTT ortholog in the model developmental system Dictyostelium discoideum, in which Ca(2+) plays a role in receptor-regulated behavior related to the aggregation process that leads to multicellular morphogenesis. The D. discoideum htt(-)-mutant failed to undergo both K(+)-facilitated chemotaxis in spatial gradients of the major chemoattractant cAMP, and chemotaxis up a spatial gradient of Ca(2+), but behaved normally in Ca(2+)-facilitated cAMP chemotaxis and Ca(2+)-dependent flow-directed motility. This was the same phenotypic profile of the null mutant of Nhel, a monovalent cation/H(+)exchanger. The htt(-)-mutant also failed to orient correctly during natural aggregation, as was the case for the Nhel mutant. Moreover, in a K(+)-based buffer the normal localization of actin was similarly defective in both htt(-) and nhe1(-) cells in a K(+)-based buffer, and the normal localization of Nhe1 was disrupted in the htt(-) mutant. These observations demonstrate that Htt and Nhel play roles in the same specific cation-facilitated behaviors and that Nhel localization is directly or indirectly regulated by Htt. Similar cation-dependent behaviors and a similar relationship between Htt and Nhe1 have not been reported for mammalian neurons and deserves investigation, especially as it may relate to Huntington׳s disease.

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

The IplA Ca2+ channel of Dictyostelium discoideum is necessary for chemotaxis mediated through Ca2+, but not through cAMP, and has a fundamental role in natural aggregation.

During aggregation of Dictyostelium discoideum, nondissipating, symmetrical, outwardly moving waves of cAMP direct cells towards aggregation centers. It has been assumed that the spatial and temporal characteristics of the front and back of each cAMP wave regulate both chemokinesis and chemotaxis. However, during the period preceding aggregation, cells acquire not only the capacity to chemotax in a spatial gradient of cAMP, but also in a spatial gradient of Ca(2+). The null mutant of the putative IplA Ca(2+) channel gene, iplA(-), undergoes normal chemotaxis in spatial gradients of cAMP and normal chemokinetic responses to increasing temporal gradients of cAMP, both generated in vitro. However, iplA(-) cells lose the capacity to undergo chemotaxis in response to a spatial gradient of Ca(2+), suggesting that IplA is either the Ca(2+) chemotaxis receptor or an essential component of the Ca(2+) chemotaxis regulatory pathway. In response to natural chemotactic waves generated by wild-type cells, the chemokinetic response of iplA(-) cells to the temporal dynamics of the cAMP wave is intact, but the capacity to reorient in the direction of the aggregation center at the onset of each wave is lost. These results suggest that transient Ca(2+) gradients formed between cells at the onset of each natural cAMP wave augment reorientation towards the aggregation center. If this hypothesis proves correct, it will provide a more complex contextual framework for interpreting D. discoideum chemotaxis.

Myosin heavy chain kinases play essential roles in Ca2+, but not cAMP, chemotaxis and the natural aggregation of Dictyostelium discoideum.

Behavioral analyses of the deletion mutants of the four known myosin II heavy chain (Mhc) kinases of Dictyostelium discoideum revealed that all play a minor role in the efficiency of basic cell motility, but none play a role in chemotaxis in a spatial gradient of cAMP generated in vitro. However, the two kinases MhckA and MhckC were essential for chemotaxis in a spatial gradient of Ca(2+), shear-induced directed movement, and reorientation in the front of waves of cAMP during natural aggregation. The phenotypes of the mutants mhckA(-) and mhckC(-) were highly similar to that of the Ca(2+) channel/receptor mutant iplA(-) and the myosin II phosphorylation mutant 3XALA, which produces constitutively unphosphorylated myosin II. These results demonstrate that IplA, MhckA and MhckC play a selective role in chemotaxis in a spatial gradient of Ca(2+), but not cAMP, and suggest that Ca(2+) chemotaxis plays a role in the orientation of cells in the front of cAMP waves during natural aggregation.

Nonsex genes in the mating type locus of Candida albicans play roles in a/α biofilm formation, including impermeability and fluconazole resistance.

The mating type locus (MTL) of Candida albicans contains the mating type genes and has, therefore, been assumed to play an exclusive role in the mating process. In mating-incompetent a/α cells, two of the mating type genes, MTLa1 and MTLα2, encode components of the a1-α2 corepressor that suppresses mating and switching. But the MTL locus of C. albicans also contains three apparently unrelated "nonsex" genes (NSGs), PIK, PAP and OBP, the first two essential for growth. Since it had been previously demonstrated that deleting either the a/α copy of the entire MTL locus, or either MTLa1 or MTLα2, affected virulence, we hypothesized that the NSGs in the MTL locus may also play a role in pathogenesis. Here by mutational analysis, it is demonstrated that both the mating type and nonsex genes in the MTL locus play roles in a/α biofilm formation, and that OBP is essential for impermeability and fluconazole resistance.

Alternative mating type configurations (a/α versus a/a or α/α) of Candida albicans result in alternative biofilms regulated by different pathways.

Similar multicellular structures can evolve within the same organism that may have different evolutionary histories, be controlled by different regulatory pathways, and play similar but nonidentical roles. In the human fungal pathogen Candida albicans, a quite extraordinary example of this has occurred. Depending upon the configuration of the mating type locus (a/α versus a/a or α/α), C. albicans forms alternative biofilms that appear similar morphologically, but exhibit dramatically different characteristics and are regulated by distinctly different signal transduction pathways. Biofilms formed by a/α cells are impermeable to molecules in the size range of 300 Da to 140 kDa, are poorly penetrated by human polymorphonuclear leukocytes (PMNs), and are resistant to antifungals. In contrast, a/a or α/α biofilms are permeable to molecules in this size range, are readily penetrated by PMNs, and are susceptible to antifungals. By mutational analyses, a/α biofilms are demonstrated to be regulated by the Ras1/cAMP pathway that includes Ras1→Cdc35→cAMP(Pde2-|)→Tpk2(Tpk1)→Efg1→Tec1→Bcr1, and a/a biofilms by the MAP kinase pathway that includes Mfα→Ste2→ (Ste4, Ste18, Cag1)→Ste11→Hst7→Cek2(Cek1)→Tec1. These observations suggest the hypothesis that while the upstream portion of the newly evolved pathway regulating a/a and α/α cell biofilms was derived intact from the upstream portion of the conserved pheromone-regulated pathway for mating, the downstream portion was derived through modification of the downstream portion of the conserved pathway for a/α biofilm formation. C. albicans therefore forms two alternative biofilms depending upon mating configuration.

Identification of genes upregulated by the transcription factor Bcr1 that are involved in impermeability, impenetrability, and drug resistance of Candida albicans a/α biofilms.

Candida albicans forms two types of biofilm, depending upon the configuration of the mating type locus. Although architecturally similar, a/α biofilms are impermeable, impenetrable, and drug resistant, whereas a/a and α/α biofilms lack these traits. The difference appears to be the result of an alternative matrix. Overexpression in a/a cells of BCR1, a master regulator of the a/α matrix, conferred impermeability, impenetrability, and drug resistance to a/a biofilms. Deletion of BCR1 in a/α cells resulted in the loss of these a/α-specific biofilm traits. Using BCR1 overexpression in a/a cells, we screened 107 genes of interest and identified 8 that were upregulated by Bcr1. When each was overexpressed in a/a biofilms, the three a/α traits were partially conferred, and when each was deleted in a/α cells, the traits were partially lost. Five of the eight genes have been implicated in iron homeostasis, and six encode proteins that are either in the wall or plasma membrane or secreted. All six possess sites for O-linked and N-linked glycosylation that, like glycosylphosphatidylinositol (GPI) anchors, can cross-link to the wall and matrix, suggesting that they may exert a structural role in conferring impermeability, impenetrability, and drug resistance, in addition to their physiological functions. The fact that in a screen of 107 genes, all 8 of the Bcr1-upregulated genes identified play a role in impermeability, impenetrability, and drug resistance suggests that the formation of the a/α matrix is highly complex and involves a larger number of genes than the initial ones identified here.

Candida albicans forms a specialized "sexual" as well as "pathogenic" biofilm.

Candida albicans forms two types of biofilm in RPMI 1640 medium, depending upon the configuration of the mating type locus. In the prevalent a/α configuration, cells form a biofilm that is impermeable, impenetrable by leukocytes, and fluconazole resistant. It is regulated by the Ras1/cyclic AMP (cAMP) pathway. In the a/a or α/α configuration, white cells form a biofilm that is architecturally similar to an a/α biofilm but, in contrast, is permeable, penetrable, and fluconazole susceptible. It is regulated by the mitogen-activated protein (MAP) kinase pathway. The MTL-homozygous biofilm has been shown to facilitate chemotropism, a step in the mating process. This has led to the hypothesis that specialized MTL-homozygous biofilms facilitate mating. If true, then MTL-homozygous biofilms should have an advantage over MTL-heterozygous biofilms in supporting mating. We have tested this prediction using a complementation strategy and show that minority opaque a/a and α/α cells seeded in MTL-homozygous biofilms mate at frequencies 1 to 2 orders of magnitude higher than in MTL-heterozygous biofilms. No difference in mating frequencies was observed between seeded patches of MTL-heterozygous and MTL-homozygous cells grown on agar at 28°C in air or 20% CO2 and at 37°C. Mating frequencies are negligible in seeded patches of both a/α and a/a cells, in contrast to seeded biofilms. Together, these results support the hypothesis that MTL-homozygous (a/a or α/α) white cells form a specialized "sexual biofilm."

Impact of environmental conditions on the form and function of Candida albicans biofilms.

Candida albicans, like other pathogens, can form complex biofilms on a variety of substrates. However, as the number of studies of gene regulation, architecture, and pathogenic traits of C. albicans biofilms has increased, so have differences in results. This suggests that depending upon the conditions employed, biofilms may vary widely, thus hampering attempts at a uniform description. Gene expression studies suggest that this may be the case. To explore this hypothesis further, we compared the architectures and traits of biofilms formed in RPMI 1640 and Spider media at 37°C in air. Biofilms formed by a/α cells in the two media differed to various degrees in cellular architecture, matrix deposition, penetrability by leukocytes, fluconazole susceptibility, and the facilitation of mating. Similar comparisons of a/a cells in the two media, however, were made difficult given that in air, although a/a cells form traditional biofilms in RPMI medium, they form polylayers composed primarily of yeast cells in Spider medium. These polylayers lack an upper hyphal/matrix region, are readily penetrated by leukocytes, are highly fluconazole susceptible, and do not facilitate mating. If, however, air is replaced with 20% CO2, a/a cells make a biofilm in Spider medium similar architecturally to that of a/α cells, which facilitates mating. A second, more cursory comparison is made between the disparate cellular architectures of a/a biofilms formed in air in RPMI and Lee's media. The results demonstrate that C. albicans forms very different types of biofilms depending upon the composition of the medium, level of CO2 in the atmosphere, and configuration of the MTL locus.

White-opaque switching in Candida albicans: cell biology, regulation, and function.

SUMMARYCandida albicans remains a major fungal pathogen colonizing humans and opportunistically invading tissue when conditions are predisposing. Part of the success of C. albicans was attributed to its capacity to form hyphae that facilitate tissue invasion. However, in 1987, a second developmental program was discovered, the "white-opaque transition," a high-frequency reversible switching system that impacted most aspects of the physiology, cell architecture, virulence, and gene expression of C. albicans. For the 15 years following the discovery of white-opaque switching, its role in the biology of C. albicans remained elusive. Then in 2002, it was discovered that in order to mate, C. albicans had to switch from white to opaque, a unique step in a yeast mating program. In 2006, three laboratories simultaneously identified a putative master switch gene, which led to a major quest to elucidate the underlying mechanisms that regulate white-opaque switching. Here, the evolving discoveries related to this complicated phenotypic transition are reviewed in a quasi-chronological order not only to provide a historical perspective but also to highlight several unique characteristics of white-opaque switching, which are fascinating and may be important to the life history and virulence of this persistent pathogen. Many of these characteristics have not been fully investigated, in many cases, leaving intriguing questions unresolved. Some of these include the function of unique channeled pimples on the opaque cell wall, the capacity to form opaque cells in the absence of the master switch gene WOR1, the formation of separate "pathogenic" and "sexual" biofilms, and the possibility that a significant portion of natural strains colonizing the lower gastrointestinal tract may be in the opaque phase. This review addresses many of these characteristics with the intent of engendering interest in resolving questions that remain unanswered.

Tec1 mediates the pheromone response of the white phenotype of Candida albicans: insights into the evolution of new signal transduction pathways.

The way in which signal transduction pathways evolve remains a mystery, primarily because we have few examples of ones that have newly evolved. There are numerous examples of how signal transduction pathways in the same organism selectively share components, most notably between the signal transduction pathways in Saccharomyces cerevisiae for the mating process, the filamentation process, cell wall integrity, ascospore formation, and osmoregulation. These examples, however, have not provided insights into how such pathways evolve. Here, through construction of an overexpression library for 107 transcription factors, and through mutational analyses, we have identified the transcription factor Tec1 as the last component of the newly evolved signal transduction pathway that regulates the pheromone response of the white cell phenotype in Candida albicans. The elucidation of this last component, Tec1, establishes a comprehensive description of the pheromone response pathway in the white cell phenotype of C. albicans, providing a unique perspective on how new signal transduction pathways may evolve. The three portions of this new regulatory pathway appear to have been derived from three different ancestral programs still functional in C. albicans. The upstream portion, including signals, receptors, the trimeric G protein complex, and the MAP kinase cascade, was derived intact from the upstream portion of the opaque pheromone response pathway of the mating process; Tec1, the transcription factor targeted by the MAP kinase pathway, was derived from a filamentation pathway; and the white-specific downstream target genes were derived from an ancestral biofilm process. The evolution of this pheromone response pathway provides a possible paradigm for how such signal transduction pathways evolve.

The "finger," a unique multicellular morphology of Candida albicans induced by CO2 and dependent upon the Ras1-cyclic AMP pathway.

Most experiments exploring the basic biology of pathogenic microbes are performed in vitro under conditions that do not usually mimic those of their host niche. Hence, developmental programs initiated by specific host cues may be missed in vitro. We have tested the effects of growing low-density agar cultures of the yeast pathogen Candida albicans in concentrations of CO(2) found in the gastrointestinal tract. It is demonstrated that in physiological concentrations of CO(2) at 37°C, yeast cells form a heretofore undescribed multicellular "finger" morphology distinct from a previously described stalk-like structure induced by high doses of UV irradiation that kills more than 99.99% of cells. The finger extends aerially, is uniform in diameter, and is visible to the naked eye, attaining lengths of 3 mm. It is composed of a basal yeast cell monolayer adhering to a semispherical crater formed in the agar and connected to a basal bulb of yeast cells at a fragile interface. The bulb extends into the long shaft. We propose that a single, centrally located hypha extending the length of the shaft forms buds at compartment junctions that serve as the source of the yeast cells in the shaft. A mutational analysis reveals finger formation is dependent upon the pathway Ras1→Cdc35→cyclic AMP (cAMP) (PDE2-|)→Tpk2→Tec1. Because of the mechanically fragile interface and the compactness of bulb and shaft, we suggest that the finger may function as a multicellular dispersal mechanism produced in host niches containing high levels of CO(2).

An unconventional myosin required for cell polarization and chemotaxis.

MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical F-actin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity.

Target specificity of the Candida albicans Efg1 regulator.

Efg1 is a central transcriptional regulator of morphogenesis and metabolism in Candida albicans. In vivo genome-wide ChIP chip and in vitro footprint analyses revealed the Efg1 recognition sequence (EGR-box) TATGCATA in the yeast growth form of this human fungal pathogen. Upstream regions of EFG1 and genes encoding transcriptional regulators of hyphal growth including TCC1, CZF1, TEC1, DEF1 and NRG1 contained EGR- and/or EGR-like boxes. Unexpectedly, after brief hyphal induction the genome-wide Efg1 binding pattern was completely altered and new binding sites of yet unknown specificity had appeared. Hyphal induction abolished Efg1 accumulation on EFG1 and TCC1 promoters and led to rapid decline of both transcripts, although the Efg1 protein persisted in cells. While EFG1 promoter activity in the yeast growth form did not depend on bound Efg1, its downregulation under hyphal induction depended on the presence of Efg1 and the protein kinase A isoform Tpk2. Deletion analyses of the EFG1 upstream region revealed that none of its resident EGR-boxes is uniquely responsible for EFG1 promoter downregulation. These results suggest different binding specificities of Efg1 in yeast growth and in hyphal induction and suggest a brief time window following hyphal induction, in which Efg1 exerts its repressive effect on target promoters.

Ca2+ chemotaxis in Dictyostelium discoideum.

Using a newly developed microfluidic chamber, we have demonstrated in vitro that Ca(2+) functions as a chemoattractant of aggregation-competent Dictyostelium discoideum amoebae, that parallel spatial gradients of cAMP and Ca(2+) are more effective than either alone, and that cAMP functions as a stronger chemoattractant than Ca(2+). Effective Ca(2+) gradients are extremely steep compared with effective cAMP gradients. This presents a paradox because there is no indication to date that steep Ca(2+) gradients are generated in aggregation territories. However, given that Ca(2+) chemotaxis is co-acquired with cAMP chemotaxis during development, we speculate on the role that Ca(2+) chemotaxis might have and the possibility that steep, transient Ca(2+) gradients are generated during natural aggregation in the interstitial regions between cells.