José Ruiz-Herrera

Polyamines, DNA Methylation, and Fungal Differentiation

Mucorales constitute a group of fungi that, because of their growth characteristics, have been used extensively in the study of cell differentiation, cell morphogenesis, and stimuli perception. We have studied the role of polyamine metabolism in the development of different Mucorales, with emphasis on Mucor and Phycomyces species. It has been observed that previous to each differentiative step, the cellular levels of the most regulated enzyme of the pathway, ornithine decarboxylase (ODC), and polyamines suffer a noticeable increase. Addition of diaminobutanone (DAB), a competitive inhibitor of ODC, blocks all the corresponding differentiative phenomena. In its presence, germinating spores fail to produce germ tubes and keep growing isodiametrically; mycelia do not sporulate but continue their vegetative growth, and yeast cells are unable to engage in a dimorphic transition without alterations in their growth rate. This differential effect of the ODC inhibitor in growth and development is apparently due to the location of ODC in at least two different cell compartments, one of which is impermeable to the drug. Inhibition of development is counteracted by putrescine and more noticeably by 5-azacytidine (5AC), a strong inhibitor of DNA methylation. Methylation levels of DNA are high in spores, and they become reduced after germination. Demethylation is inhibited by hydroxyurea, which blocks DNA replication, and by DAB. The effect of the latter is reversed by 5AC. These results suggest a relationship between polyamines and DNA methylation. Analysis of metallothioneine gene (CUP) behavior and expression during spore germination has confirmed this hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the dimorphic transition of Candida albicans by the ornithine decarboxylase inhibitor 1,4-diaminobutanone: Alterations in the glycoprotein composition of the cell wall

Hyphal development in Candida albicans was selectively blocked by the ornithine decarboxylase competitive inhibitor 1,4-diaminobutanone (DAB). Inhibition of hyphal development required DAB during both yeast inoculum growth and subsequent incubation at 37 degrees C to induce mycelial growth. This effect was not due to general growth inhibition since DAB did not inhibit yeast growth, and reduced protein synthesis by 30% at most. Moreover, protein synthesis was unaffected by DAB when cells were pre-grown in drug-containing media. Since DAB inhibited dimorphic transition at 37 degrees C, morphology- and temperature-dependent protein synthesis could be distinguished. DAB stimulated the synthesis of several yeast wall-proteins, irrespective of morphology or growth temperature, and two at 37 degrees C only, but it inhibited the synthesis of a single mycelial-specific glycoprotein species.

Biogenesis of the Fungal Cell Wall

Cell walls play essential roles in growth, development, and in interactions of fungi with the environment and with other cells. Besides its primary protective role in shielding the cell against osmotic, chemical, and biological harm, the wall is involved in many other functions including morphogenesis, and some activities that may be denominated as “social”, such as morphological responses, antigenic expression, adhesion, and cell-cell interaction (Peberdy 1990; Ruiz-Herrera 1992; Sentandreu et al. 1991). There are many data supporting the idea that temporal and spatial regulation of wall polymer synthesis and assembly are critical for the properties of the walls, which thus do not exclusively depend on their chemical composition, but also on the way that different polymers interact.

Light response inPhycomyces blakesleeanus: Evidence for roles of chitin biosynthesis and breakdown

Abstract Chitin synthetase activity, both basal and zymogenic, from Phycomyces sporangiophores was stimulated by light in vitro and in vivo . A mad B mutant did not display these activations, whereas in a mad E mutant only chitin synthetase zymogen was increased by illumination in vivo . Light also produced a transient alteration in cell wall structure at the apical region of the sporangiophore revealed by accessibility of chitin to binding by wheat germ agglutinin and by an increased limited breakage of chitin microfibrils. This last response was absent in both mad B and mad E mutants. Accordingly, it is suggested that the light growth response in the sporangiophore from Phycomyces is due to a transient softening of the cell wall at the growing region followed by an elongation due to the turgor pressure of the cell and an enhanced chitin biosynthesis by the apically localized chitin synthetase which restores normal strength to the cell wall. A hypothetical scheme to account for these results is presented.

Chitosomes from the wall-less “slime” mutant of Neurospora crassa

Cell-free extracts from the wall-less slime mutant of Neurospora crassa and the mycelium of wild type exhibit similar chitin synthetase properties in specific activity, zymogenicity and a preferential intracellular localization of chitosomes. The yield of chitosomal chitin synthetase from sline cells was essentially the same irrespective of cell breakage procedure (osmotic lysis or ballistic disruption) —an indication that chitosomes are not fragments of larger membranes produced by harsh (ballistic) disruption procedures. The plasma membrane fraction, isolated from slime cells treated with concanavalin A, contained only a minute portion of the total chitin synthetase of the fungus. Most of the activity was in the cytoplasmic fraction; isopycnic sedimentation of this fraction on a sucrose gradient yielded a sharp band of chitosomes with a buoyant density=1.125 g/ cm3. Approximately 76% of the total chitin synthetase activity of the slime mutant was recovered in the chitosome band. Because of their low density, chitosomes could be cleanly separated from the rest of the membranous organelles of the fungus. Apparently, the lack of a cell wall in the slime mutant is not due to the absence of either chitosomes or zymogenic chitin synthetase.

Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin

Levels of basal chitin synthetase in cell-free extracts from Phycomyces blakesleeanus were reduced by breakage of cells in the presence of EDTA or EGTA. Addition of Ca2+ to these extracts activated chitin synthetase. Maximal activation was obtained after 2 h at a Ca2+ concentration of 2–5 mM. Activation by calcium was not reduced by any protease inhibitor tested but benzamidine, whereas the weak proteolytic activity of the extracts was inhibited by antipain. Larger levels of chitin synthetase activation were obtained by the simultaneous addition of calcium and calmodulin in most, but not all extracts. This further activation by calmodulin was prevented by TFP. ATP or cAMP did not stimulate activation by calcium or calcium-calmodulin.

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone.

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.

Regulation of ornithine decarboxylase activity in Mucor bacilliformis and Mucor rouxii

Abstract Ornithine decarboxylase (ODC) from Mucor bacilliformis and Mucor rouxii was studied. Enzymatic activity was maximal at pH 7.2–7.4 and at 30°C. The K m was 0.17 m M for the M. bacilliformis enzyme. Putrescine was a competitive inhibitor of ODC with a K i of 2–3 m M . Enzymatic activity was undetectable in sporangiospores but increased rapidly during the first stages of spore swelling, reaching the highest levels during germ tube or bud emergence, and then decreased. Incubation at 30°C inhibited spore germination in M. bacilliformis and prevented development of ODC activity. More ODC activity was present in mycelial than in yeast cells. Morphological transition of yeast cells into hyphae by an anaerobic-aerobic shift induced a rapid and transient increase in ODC activity. Similar results were obtained when the morphogenetic transformation of M. rouxii was induced by CO 2 elimination in an anaerobic environment. Transfer of mycelial cells to anaerobiosis resulted in a rapid decrease in enzyme activity. Changes in ODC activity were accompanied by a change in the pool of polyamines. The possible role of ODC in growth and cell differentiation in Mucor is discussed.

Effect of ornithine decarboxylase inhibitors on the germination of sporangiospores of mucorales

Of several known inhibitors of ornithine decarboxylase activity, only 1,4-diamino-2-butanone (DAB) inhibited spore germination of Mucor rouxii. Drug-exposed cells went through the phase of spherical growth attaining huge cell volumes, but were unable to germinate. The same phenomenon occurred irrespective of the growth medium in several species of Mucorales. DAB barely inhibited the biosynthesis of proteins and DNA during the phase of spherical growth. Higher levels of inhibition were observed at later growth periods. The addition of DAB to the growth medium decreased the cellular levels of polyamines. Removal of DAB or addition of putrescine reversed the inhibitory effect. Cycloheximide and actinomycin D completely blocked the reversal of the inhibition of germination after DAB removal or addition of putrescine, whereas hydroxyurea gave variable intermediate values of inhibition. It is suggested that the DAB effect can be interpreted as a requirement of high levels of polyamines for the change of growth patterns from spherical to apical in the fungi studied.

Alterations in the biosynthesis of chitin and glucan in the slime mutant ofNeurospora crassa

Abstract The slime mutant of Neurospora crassa lacks both chitin and insolubleβ(1 → 3)glucans either cellbound or secreted to the medium. Failure to synthesize both polymers is not due to deficiencies in the precursor sugar nucleotides, UDP-GlcNAc and UDP-Glc, since these are present in higher amounts in the mutant than in the wild type. The slime mutant also contains higher levels of chitin synthetase than the wild type, but lacks detectable glucan synthetase activity. an os-1 mutant grown in hypertonic medium contained reduced amounts of chitin and glucan, but its glucan and chitin synthetase activities were nearly normal. It is concluded that the absence of a cell wall in the slime mutant is due to a mutational event(s) which affects the mechanism of activation or delivery of chitin synthetase to the cell surface and the formation of a functionalβ(1 → 3)glucan synthetase. This mutational event must be different in the os-1 strain.