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Disordered ultrastructure in lignin-peroxidase-secreting hyphae of the white-rot fungus Phanerochaete chrysosporium.

The practice of exposing liquid cultures of the white-rot fungus Phanerochaete chrysosporium to a pure oxygen atmosphere under conditions of nutrient starvation has been widely adopted to induce lignin peroxidase (LiP) synthesis. Transmission electron microscopy was used to examine hyphal cells of carbon-limited cultures that had been exposed to an atmosphere of pure oxygen, and revealed evidence of a major loss in organization of cellular ultrastructure, which may be attributed to oxygen toxicity. Under some conditions (continuous agitation in air with cellulose as the carbon source) cultures will produce LiP without needing to be exposed to a pure oxygen atmosphere. A similar major loss of cellular ultrastructure was found in hyphal cells from such cultures upon examination. Investigation of the levels of H2O2, catalase and carbonyl content of intracellular proteins suggests that the latter cultures developed a hyperoxidant state because the rate of supply of carbon from cellulose hydrolysis was insufficient for oxygen homeostasis. The association of LiP with these cultures and with those exposed to an atmosphere of pure oxygen infers that LiP may be triggered in response to oxidant stress.

Carbon↗

The effect of 5-fluorocytosine on the blastospores and hyphae of Candida albicans.

In Candida albicans the continued increase in dry weight, in cell volume and in hyphal length during 5FC treatment is mainly due to increased amount of carbohydrate despite the decreased amounts of nucleic acids. Incorporation studies with 32PO4 (for RNA) in C. albicans and with 3H-thymidine-monophosphate (for DNA) in a thymidine-utilising strain of Saccharomyces cerevisiae have shown that the decreased amounts of nucleic acids were due to an inhibition of synthesis of RNA and DNA by 5FC. Nuclear-staining techniques on the hyphal phase of C. albicans showed that 5FC inhibits nuclear division. The changes in amounts of protein during 5FC treatment do not wholly explain the changes in cell size although 14C-histidine incorporation experiments showed that protein synthesis continued in the presence of 5FC. 14C-glucose incorporation in the presence of 5FC showed an initial accelerated synthesis of carbohydrate with a maintained level of synthesis after 16 h. This abnormal pattern of synthesis correlates with the increase in amount of carbohydrate and in cell size and hyphal elongation. 5FC inhibits DNA synthesis, and all manifestations of unbalanced growth that culminate in the cell volume changes appear to be a consequence of that inhibition.

Candida albicans↗

Flocculation of hyphae is associated with a deletion in the putative CaHK1 two-component histidine kinase gene from Candida albicans.

In Candida albicans, three putative histidine kinase genes have been described thus far, including CaSLN1, CaNIK1/COS1 and CaHK1. The encoded proteins for C. albicans, CaSln1p and CaNik1p, which are similar to Sln1p from Saccharomyces cerevisiae and Nik-1 from Neurospora crassa, seem to function in osmoregulation and morphogenesis, respectively. Recently, the isolation of CaHK1, a putative histidine kinase gene from C. albicans has been reported. In addition to the histidine and aspartyl domains located at its C-terminus as previously described, it is shown here that the N-terminal domain of Cahk1p contains a P-loop motif and a sequence which shows significant homology with the seven C-terminal domains of serine/threonine kinases. The Ser/Thr-homologous domains of Cahk1p could, in fact, correspond to its sensor sequence. CaHK1 has been mapped to chromosome 2 and gene deletion studies were undertaken to understand its function. Deltacahk1 mutants are phenotypically different from any other histidine kinase mutants thus far described either in C. albicans or in any other yeast or filamentous fungus. This study demonstrates that deltacahk1 mutants flocculate extensively in a gene-dosage-dependent manner under conditions which induce germ-tube formation, such as growth in medium 199 (pH 7.5). The flocculation occurs by an interaction along the hyphal surfaces, probably because of the altered expression of one or more hyphal-cell-surface components in the deltacahk1 mutants. These results indicate that CaHK1 could be involved in regulating their expression.

Amino Acid Sequence↗

Myceligenerans xiligouense gen. nov., sp. nov., a novel hyphae-forming member of the family Promicromonosporaceae.

Strain XLG9A10.2T was isolated from an alkaline salt marsh soil in western China. 16S rRNA gene sequence analysis indicated that strain XLG9A10.2T constitutes a distinct lineage within the family Promicromonosporaceae, sharing 94.8-95.1% gene similarity with members of the genus Promicromonospora and 94.4-95.7% similarity with those of Xylanimonas and related genera. The general colony and cell morphology of strain XLG9A10.2T is similar to that of members of Promicromonospora, but differs from members of the genus Xylanimonas in forming a well-developed branching mycelium and production of coccoid spores. Strain XLG9A10.2T shows the peptidoglycan type A4alpha (L-lys<--L-thr<--D-Glu), contains glucose, mannose and galactose as whole cell sugars and has MK-9(H4) and MK-9(H6) as major menaquinones, while phospholipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, three unidentified phospholipids and one unidentified glycolipid. The DNA base composition is 71.9 mol% G+C. On the basis of morphological, chemotaxonomic, metabolic and phylogenetic differences from other species of Promicromonosporaceae, a new genus and species, Myceligenerans xiligouense gen. nov., sp. nov., is proposed. The type strain is XLG9A10.2T (=DSM 15700T=CGMCC 1.3458T.)

Actinomycetales↗

Inhibition of RNA synthesis in Neurospora crassa hyphae treated with picolinic acid.

Picolinic acid, a metal-chelating agent, blocks the nuclei of germinating conidia of Neurospora crassa in the G1 phase. The addition of picolinic acid to exponentially growing cultures of N. crassa in glucose medium causes an immediate inhibition of growth. Concentrations higher than 50 mM completely inhibit growth of mycelia, whereas lower concentrations (20-30 mM) give only a partial inhibition. Picolinic acid, added at the concentration of 20 mM, immediately blocks the accumulation of stable RNA, while protein accumulation continues at an unchanged rate for at least 30 min and only thereafter is slightly inhibited. DNA accumulation rapidly slows down after the addition of the drug. The level of polyadenylated RNA decreases quickly after the addition of picolinic acid, and the rate of its synthesis also appears to be inhibited. Picolinic acid greatly affects the metabolism of stable RNA in N. crassa: both processing and transcription of rRNA are sequentially inhibited with a moderate accumulation of precursor rRNA. It is concluded that the effects of picolinic acid on the nuclear division cycle in Neurospora may be related to the requirement of specific gene products to enter the S phase, whose synthesis is inhibited following the reduction of polyadenylated RNA synthesis.

Centrifugation, Density Gradient↗

Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) contains a cluster of putative isoprenoid and hopanoid biosynthetic genes. The strain does not produce the pentacyclic hopanoids in liquid culture but produces them on solid medium when sporulating. Mutants defective in the formation of aerial mycelium and spores (bld), with the exception of bldB, do not synthesize hopanoids, whereas mutants, which form aerial mycelium but no spores (whi), do. The membrane condensing hopanoids possibly may alleviate stress in aerial mycelium by diminishing water permeability across the membrane.

Culture Media↗

Comparison of in vitro antifungal susceptibilities of conidia and hyphae of filamentous fungi.

The MICs and minimum fungicidal concentrations (MFCs) of amphotericin B, fluconazole, ketoconazole, flucytosine, miconazole, and itraconazole for 12 isolates of filamentous opportunistic fungi (Scopulariopsis sp., Paecilomyces sp., Cladosporium spp., and Cladophialophora sp.) were determined by a broth microdilution method with hyphal and conidial inocula. With hyphal inocula MICs and MFCs were practically always substantially higher. Only 25% of the 60 MIC comparisons showed discrepancies of twofold or less, while the remaining comparisons showed much larger differences.

Antifungal Agents↗

The Kip3-like kinesin KipB moves along microtubules and determines spindle position during synchronized mitoses in Aspergillus nidulans hyphae.

Kinesins are motor proteins which are classified into 11 different families. We identified 11 kinesin-like proteins in the genome of the filamentous fungus Aspergillus nidulans. Relatedness analyses based on the motor domains grouped them into nine families. In this paper, we characterize KipB as a member of the Kip3 family of microtubule depolymerases. The closest homologues of KipB are Saccharomyces cerevisiae Kip3 and Schizosaccharomyces pombe Klp5 and Klp6, but sequence similarities outside the motor domain are very low. A disruption of kipB demonstrated that it is not essential for vegetative growth. kipB mutant strains were resistant to high concentrations of the microtubule-destabilizing drug benomyl, suggesting that KipB destabilizes microtubules. kipB mutations caused a failure of spindle positioning in the cell, a delay in mitotic progression, an increased number of bent mitotic spindles, and a decrease in the depolymerization of cytoplasmic microtubules during interphase and mitosis. Meiosis and ascospore formation were not affected. Disruption of the kipB gene was synthetically lethal in combination with the temperature-sensitive mitotic kinesin motor mutation bimC4, suggesting an important but redundant role of KipB in mitosis. KipB localized to cytoplasmic, astral, and mitotic microtubules in a discontinuous pattern, and spots of green fluorescent protein moved along microtubules toward the plus ends.

Amino Acid Sequence↗