Terry W. Hill

Molecular characterization and influence on fungal development of ALP2, a novel serine proteinase from Aspergillus fumigatus.

A novel subtilisin-related serine proteinase (ALP2) [EC 3.4.21.48] with a broad range of activity between pH 4.5 and 11.0 was released from a cell wall fraction of Aspergillus fumigatus by an alkaline pH shift. The enzyme which was not detected in the culture supernatant was partially purified by phenylbutylamine agarose chromatography. The N-terminal sequence revealed that ALP2 is the same protein identified as the major allergen of A. fumigatus in patients suffering from extrinsic bronchial asthma (Shen et al. 1999, Int. Arch. Allergy Immunol. 119, 259-264). Based on this N-terminal sequence and on a conserved region of fungal subtilisins, a specific PCR probe was generated and the ALP2 genomic and cDNA were isolated from corresponding phage libraries. ALP2 shares a 49% identity with the vacuolar proteinase B (PrB) of Saccharomyces cerevisiae. In addition there is a 78% identity with PEPC, a serine proteinase which has been described in Aspergillus niger. Targeted disruption of the ALP2-encoding gene resulted in a slightly decreased speed of vegetative growth and in a more than 80% reduction of sporulation in the alp2-negative mutants, correlated with an approximately 50% reduction of the median diameter of conidiophore vesicles. The requirement of ALP2 for regular sporulation, in addition to its role in allergic asthma, raises further interest in cellular proteinases in respect to morphogenesis and pathogenesis in A. fumigatus.

Two GDP-mannose transporters contribute to hyphal form and cell wall integrity in Aspergillus nidulans

In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans that show hypersensitivity to the chitin-binding agent Calcofluor White (CFW). Affected loci are designated cal loci. The phenotype of one of these alleles, calI11, also includes shortened hyphal compartments and increased density of branching in the absence of CFW, as well as reduced staining of cell walls by the lectin FITC-Concanavalin A (ConA), which has strong binding affinity for mannosyl residues. We have identified two A. nidulans genes (AN8848.3 and AN9298.3, designated gmtA and gmtB, respectively) that complement all aspects of the phenotype. Both genes show strong sequence similarity to GDP-mannose transporters (GMTs) of Saccharomyces and other yeasts. Sequencing of gmtA from the calI11 mutant strain reveals a G to C mutation at position 943, resulting in a predicted alanine to proline substitution at amino acid position 315 within a region that is highly conserved among other fungi. No mutations were observed in the mutant strains allele of gmtB. Meiotic mapping demonstrated a recombination frequency of under 1 % between the calI locus and the phenA locus (located approximately 9.5 kb from AN8848.3), confirming that gmtA and calI are identical. A GmtA-GFP chimera exhibits a punctate distribution pattern, consistent with that shown by putative Golgi markers in A. nidulans. However, this distribution did not overlap with that of the putative Golgi equivalent marker CopA-monomeric red fluorescent protein (mRFP), which may indicate that the physically separated Golgi-equivalent organelles of A. nidulans represent physiologically distinct counterparts of the stacked cisternae of plants and animals. These findings demonstrate that gmtA and gmtB play roles in cell wall metabolism in A. nidulans similar to those previously reported for GMTs in yeasts.

Isolation and characterization of an endo-(1,4)-β-glucanase secreted by Achlya ambisexualis

Models of wall loosening in fungi and other walled eukaryotes require the action of proteins able to reduce the degree of linkage between components of the wall. In the oomycete Achlya ambisexualis, such a role has been proposed for a suite of endoglucanases that are secreted during branching and during the measurable wall softening associated with osmotic stress. We report here the isolation and characterization of one of these isoenzymes. The enzyme has a molecular weight of 32 kDa, a pH optimum of 6.75, a pI of 4.5, and a temperature optimum of 35 C. It is partially inhibited by sulfhydryl-binding reagents and completely inhibited by the tryptophan-binding reagent NBS. The enzyme has an endohydrolytic mode of action with substrate specificity towards glucans that contain β-(1,4) linkages, either alone (carboxymethyl cellulose) or as mixed linkage (1,4–1,3)-β-glucans (e.g., Avena glucan). It does not, however, degrade amorphous insoluble (phosphoric acid swollen) cellulose. Most significantly, the enzyme can also hydrolyze linkages in an Achlya cell wall fraction previously shown to consist of a mixed-linkage (1,4–1,3)-β-glucan. This property is consistent with the long-standing hypothesis that the branching-related endoglucanases of oomycetes play a role in cell wall loosening.

GDP-mannose transporter paralogues play distinct roles in polarized growth of Aspergillus nidulans

GDP-mannose transporters (GMT) carry GDP-mannose nucleotide sugars from the cytosol across the Golgi apparatus membrane for use as substrates in protein glycosylation in plants, animals and fungi. Genomes of some fungal species, such as the yeast Saccharomyces cerevisiae, contain only one gene encoding a GMT, while others, including Aspergillus nidulans, contain two (gmtA and gmtB). We previously showed that cell wall integrity and normal hyphal morphogenesis in A. nidulans depend upon the function of GmtA and that GmtA localizes to a Golgi-like compartment. Cells bearing the calI11 mutation in gmtA also have reduced cell surface mannosylation. Here we show that GmtB colocalizes with GmtA, suggesting that the role of GmtB is similar to that of GmtA, although the respective transcript levels differ during spore germination and early development. Transcript levels of gmtB are high in ungerminated spores and remain so throughout the first 16 h of germination. In contrast, transcript levels of gmtA are negligible in ungerminated spores but increase to levels comparable to those of gmtB during germination. These observations suggest that although GmtA and GmtB reside within the same subcellular compartments, they nevertheless perform distinct functions at different stages of development.

Mutations in proteins of the Conserved Oligomeric Golgi Complex affect polarity, cell wall structure, and glycosylation in the filamentous fungus Aspergillus nidulans.

We have described two Aspergillus nidulans gene mutations, designated podB1 (polarity defective) and swoP1 (swollen cell), which cause temperature-sensitive defects during polarization. Mutant strains also displayed unevenness and abnormal thickness of cell walls. Un-polarized or poorly-polarized mutant cells were capable of establishing normal polarity after a shift to a permissive temperature, and mutant hyphae shifted from permissive to restrictive temperature show wall and polarity abnormalities in subsequent growth. The mutated genes (podB=AN8226.3; swoP=AN7462.3) were identified as homologues of COG2 and COG4, respectively, each predicted to encode a subunit of the multi-protein COG (Conserved Oligomeric Golgi) Complex involved in retrograde vesicle trafficking in the Golgi apparatus. Down-regulation of COG2 or COG4 resulted in abnormal polarization and cell wall staining. The GFP-tagged COG2 and COG4 homologues displayed punctate, Golgi-like localization. Lectin-blotting indicated that protein glycosylation was altered in the mutant strains compared to the wild type. A multicopy expression experiment showed evidence for functional interactions between the homologues COG2 and COG4 as well as between COG2 and COG3. To date, this work is the first regarding a functional role of the COG proteins in the development of a filamentous fungus.

Association of UDPG transferase activity with cell walls of Achlya ambisexualis

Fungal cell wall growth has been postulated to involve the cooperative action of both wall-hydrolyzing and wall-synthesizing enzymes (Park and Robinson, 1966; Gooday and Trinci, 1980). Investigations of cell wall morphogenesis in Achlya have dealt primarily with hydrolytic events, particularly those mediated by endo-l,4-D-glucanases (Thomas and Mullins, 1967, 1969; Hill and Mullins, 1979, 1980). Much less attention has been devoted to wall-synthesizing enzymes in Achlya and other cellulosic fungi because the activity of glucan-synthesizing enzymes is difficult to demonstrate in vitro, and the low level of activity makes characterization of the products uncertain. In a previous investigation, uridinediphosphoglucose (UDPG) transferase (EC 2.1.4.12) was demonstrated at low activity in association with subcellular particles that also exhibited cellulase activity (Hill and Mullins, 1980). Since these particles were similar to apical vesicles this association suggests that UDPG transferase in Achlya may be involved in cell wall synthesis. In the present paper, activity of UDPG transferase is demonstrated in cell walls of Achlya, and the solubility properties of the in vitro reaction products are described. Achlya ambisexualis Raper strain E87 was cultured for 48 h as described previously (Mullins, 1973). UDPG tranferase activity was assayed by a modification of the techniques of Ray et al. (1969) and Shore and Maclachlan (1975). The reaction mixture contained (mM): UDPG, 0.24; cellobiose, 5; MgCl2, 11; dithiothreitol (DTT), 1.7; pH 5.8 sodium phosphate buffer, 67; and incubation was 20 min at 23 C. The UDPG was glucose-uL-14C (250 utCi/Aumole, New England Nuclear) and as used gave 7.3 x 10-8 moles with 119 nCi of radioactivity. Products were recovered by centrifugation of 70% ethanol-insoluble material after addition of 30 mg of powdered Whatman cellulose as a carrier. To determine the distribution of transferase activity between wall and protoplasm fractions, mycelia were homogenized by ultrasonication at 5-10 C in a buffered homogenizing solution containing 20% w/w sucrose, 10 mM DTT, and 20 mM tris HCl buffer, pH 7.6. Hyphal fragments were sedimented by centrifugation, then resuspended, and the entire process repeated three times. The supernatant fractions were pooled and the final sediment, when examined by phase contrast microscopy, consisted of wall fragments without apparent cytoplasmic contamination. UDPG transferase activity was assayed in each fraction and protein was estimated with the Branford (1976) Bio-Rad assay. Results are displayed in TABLE I. Although less transferase activity was found in the wall fraction than 851

Two amino acid sequences direct Aspergillus nidulans protein kinase C (PkcA) localization to hyphal apices and septation sites.

The Aspergillus nidulans ortholog of protein kinase C (pkcA) is involved in the organism’s putative cell wall integrity (CWI) pathway, and PkcA also is highly localized at growing tips and forming septa. In the present work we identify the regions within PkcA that are responsible for its localization to hyphal tips and septation sites. To this end, we used serially truncated pkcA constructs and expressed them as green fluorescent protein (GFP) chimeras and identified two regions that direct PkcA localization. The first region is a 10 amino-acid sequence near the carboxyl end of the C2 domain that is required for localization to hyphal tips. Proteins containing this sequence also localize to septation sites. A second region between C2 and C1B (encompassing C1A) is sufficient for localization to septation sites but not to hyphal tips. We also report that localization to hyphal tips and septation sites alone is not sufficient for truncated constructs to complement hypersensitivity to the cell wall compromising agent calcofluor white in a strain bearing a mutation in the pkcA gene. Taken together, these results suggest that localization and stress response might be independent.

Efficient high-volume cleaning of Aspergillus nidulans cleistothecia using bare fingers.

Even seasoned workers find it tedious and sometimes frustrating to remove Hülle cells and stray conidia from Aspergillus cleistothecia by rolling them with forceps across an agar surface, particularly when large numbers must be cleaned. It can be even more challenging to teach the skill to others, especially to a whole class of easily discouraged undergraduates, who may be seeing their first high-mag image of forceps tips at the same time as their first view of a cleistothecium. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol50/iss1/2 4 Fungal Genetics Newsletter Efficient high-volume cleaning of Aspergillus nidulans cleistothecia using bare fingers. Terry W. Hill, Darlene M. Loprete, Jared A, Castagna, and Samuel O. Weems. Department of Biology and Department of Chemistry, Rhodes College, Memphis, TN 38112 USA. Even seasoned workers find it tedious and sometimes frustrating to remove Hülle cells and stray conidia from Aspergillus cleistothecia by rolling them with forceps across an agar surface, particularly when large numbers must be cleaned. It can be even more challenging to teach the skill to others, especially to a whole class of easily discouraged undergraduates, who may be seeing their first high-mag image of forceps tips at the same time as their first view of a cleistothecium. The tips of forceps are sharp and rough, and the hands that wield them often shaky. A peridium is no match for an ill-aimed poke or slash. We find, however, that cleistothecia can be cleaned very rapidly, with reduced breakage, and with minimal contamination by doing away with forceps altogether and using instead the tools that nature gave us “at our fingertips”. Indeed they are our fingertips themselves. The method is simply to roll cleistothecia briefly and firmly around the surface of a 4% agar plate (with or without diatomaceous earth per Kaminskyj and Hamer, 1996, Fungal Genetics Newsletter 43:71) beneath a well-cleaned fingertip. A few seconds’ rubbing in a circle about an inch in diameter is all that is required. The pressure of the finger is spread evenly, and the cleistothecia only rarely break. For increased efficiency, several cleistothecia can be rubbed at once beneath a single fingertip. Soap-andwater washing, followed by two or three 10-second immersions of the finger in 95% alcohol, with Kimwipe-drying after each immersion, is sufficient in our experience to reduce levels of bacterial contamination to no more than those observed when using flamed forceps – i.e., essentially none. Published by New Prairie Press, 2017