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The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4 allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression of SIT4 leads to an elevation in resistance to paromomycin and to lesser extent tolerance to sorbic acid, but it has no detectable effect on resistance to 4-NQO. These observations suggest that the Sit4 protein phosphatase has a broad role in modulating multidrug resistance in K. lactis. Expression or activity of a membrane transporter specific for paromomycin and the ABC pumps responsible for 4-NQO and sorbic acid would be positively regulated by Sit4p. In contrast, the function of a Pdr5-type transporter responsible for ketoconazole and econazole extrusion, and probably also for efflux of oligomycin and antimycin, is likely to be negatively regulated by the phosphatase. Drug resistance of sit4 mutants was shown to be mediated by ABC transporters as efflux of the anionic fluorescent dye rhodamine 6G, a substrate for the Pdr5-type pump, is markedly increased in sit4 mutants in an energy-dependent and FK506-sensitive manner.

Purified mitochondria from the petite positive yeast Torulopsis glabrata contain a circular deoxyribonucleic acid (DNA) with a length of 6 mum and a buoyant density of 1.686 g/cm3. This DNA is absent from ethidium bromide induced respiratory-deficient mutants.

The role of specialized regions of insect rectal papillae in the regulation of water and ion uptake is well documented. Although the apparatus for active uptake of water or ions is located in various cell membranes, the absorbed molecules must first pass through the cuticle which lines the rectal epithelium. Most cuticle (e.g. abdominal) has been shown to be permeable only to molecules soluble in wax, and to be impermeable to water and ions. Obviously if such cuticle lined the rectum, absorption of water and ions would be severely restricted. The present freeze-fracture and lanthanum tracer study was undertaken to investigate in more detail both the morphological features of the rectal papillae cuticle which could be responsible for its anomalous permeability and the various cell membranes involved in this transport. It has been suggested from permeability studies that the anomalous permeability of rectal papillae cuticle could be due to the lack of a complete wax layer over the surface of the rectal cuticle. The present study strongly supports this suggestion. Thus, the freeze-fracture micrographs have shown that a surface layer of the cuticle reacts during fracturing like a lipid bilayer. However, in rectal papilla cuticle this surface bilayer is interrupted at each epicuticular depression by areas of different fracturing behaviour. These discontinuities in the surface bilayer probably allow the rectal contents to contact directly the true cuticular matrix. They could, therefore, explain the ease with which water and ions penetrate the rectal cuticle and so gain access to the underlying epithelial cells. Although similar discontinuities are present on some of the rectal cuticle surface external to the rectal papillae, they appear to be filled in by plugs of lipid-like material. The lateral plasma membranes of the rectal papillae cells are generally considered to be the main site of active transport. The present lanthanum tracer and freeze-fracture study has shown that the lateral plasma membranes contain 3 distinct differentiations. Septate junctions are present at the apical and basal surfaces of the epithelial layer; a further membrane differentiation is found adjacent to the septate junctions; and thirdly, an array of short, variable length, non-anastomosing linear structures covers most of the lateral plasma membrane surface. These latter structures, unlike known types of cell junctions do not show equivalent arrays in apposing membranes even when the lateral plasma membranes of adjacent cells are closely apposed. The possible function of these structures is discussed.

Mucor genevensiswas grown in both glucose-limited and glucose-excess continuous cultures over a range of dissolved oxygen concentrations (<0.1 to 25 μM) to determine the effects of glucose and the influence of metabolic mode (fermentative versus oxidative) on dimorphic transformations in this organism. The extent of differentiation between yeast and mycelial phases has been correlated with physiological and biochemical parameters of the cultures. Under glucose limitation, oxidative metabolism increased as the dissolved oxygen concentration increased, and this paralleled the increase in the proportion of the mycelial phase in the cultures. Filamentous growth and oxidative metabolism were both inhibited by glucose even though mitochondrial development was only slightly repressed. However, the presence of chloramphenicol in glucose-limited aerobic cultures inhibited mitochondrial respiratory development but did not induce yeast-like growth, indicating that oxidative metabolism is not essential for mycelial development. Once mycelial cultures had been established under aerobic, glucose-limited conditions, subsequent reversal to anaerobic conditions or treatment with chloramphenicol caused only a limited reversal (<35%) to the yeast-like form. Glucose, however, induced a complete reversion to yeast-like form. It is concluded that glucose is the most important single culture factor determining the morphological status of M. genevensis; mitochondrial development and the functional oxidative capacities of the cell appear to be less important factors in the differentiation process.

Growth of Mucor genevensis, a facultatively anaerobic dimorphic mold, in high concentrations of chloramphenicol (4 mg/ml) leads to increased numbers of yeast-like cells and small club-like mycelial forms. This change in morphology is accompanied by a threefold increase in the mass doubling time, the loss of cyanide-sensitive respiration, and the development of cyanide-insensitive respiration. Associated with these changes is the absence of cytochromes aa3and band the inability of the organism to utilize ethanol; in addition, mitochondria appear more numerous and have less internal membrane. A further inhibitory action of the antibiotic, other than eliminating functional mitochondria, appears likely since microaerobic cultures which lack respiratory ability have twice the mass doubling time in the presence of the drug. Although a small inhibition of amino acid incorporation by cytoplasmic ribosomes is found with a high chloramphenicol concentration, it is insufficient to account for the effect on growth of the microaerobic culture. The nature of this additional effect of chloramphenicol remains to be determined, but it has been shown that increasing the glucose concentration can partially reverse this action of the antibiotic. The effect of the drug on the morphology of the organism is not as dramatic as that of phenethyl alcohol in producing yeast-like forms. However, in view of the action of chloramphenicol in eliminating functional mitochondria in M. genevensisthe suggestion that phenethyl alcohol exerts its effect in promoting yeast-like morphology by uncoupling oxidative phosphorylation should be re-examined.

Fatty acid and sterol content and composition were determined for the dimorphic mold, Mucor genevensis, grown under a variety of experimental conditions. Fatty acids account for 6 to 9% of the dry weight of aerobically grown mycelium, and 70 to 80% of these are unsaturated. The organism contains γ-linolenic acid which is characteristic for Phycomycetes, and in sporangiospores this compound represents 40% of the total fatty acids. Of the sterols found in mycelium, 80% is ergosterol, and stigmasterol was positively identified as one of the minor components. In anaerobically grown yeastlike cells, sterol content is less than 10% of the level found in aerobically grown cells, and fatty acids amount to less than 2% of the dry weight. These fatty acids are predominantly short chain and less than 10% are unsaturated. Yeastlike cells obtained under aerobic conditions by growth in the presence of phenethyl alcohol have fatty acid and sterol compositions characteristic of aerobically grown mycelium. It is concluded that the dimorphology of the organism is not directly related to lipid composition.

The addition of nitrate to cultures of Spirillum itersoniiincubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 × g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome cwas increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome crose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome cin response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome cdoes not require the presence of a functional nitrate reductase.

Mitomycin C and ultraviolet light were found to induce the formation of microcysts in Spirillum itersonii. These forms, as well as spontaneously occurring microcysts in this species, were found to contain phage tail parts, rhapidosomes, and a granular substance not seen in normal cells. It is suggested that microcysts are formed as the result of the induction of a defective phage. The production of phage lysozyme within the cell could lead to the formation of spherical forms as the cells lose their structural mucopeptide layer. Complete virus particles were not seen, nor was any biological activity demonstrated when the induced cultures were tested against two other strains of S. itersonii. The other strains of this bacterium also formed microcysts and phage tail parts when induced with mitomycin. Attempts to isolate an organism lacking the defective phage have been unsuccessful.

Spirillum itersoniicontains b- and c-type cytochromes as well as a carbon monoxide-binding pigment of the cytochrome otype. Synthesis of cytochromes band cis increased by about two- and fourfold, respectively, when cells are transferred from high to low aeration. The increased concentration of cytochrome is not accompanied by an increase in the respiration rate of the cells. Both cytochrome band cytochrome care located in the particulate fraction of cells grown under high or low aeration, and both pigments are fully reducible by succinate. No evidence was found for the accumulation of the protein component of either cytochrome when synthesis of the prosthetic group was limited by iron deficiency, nor did heme or precursors accumulate when protein synthesis was prevented. It was therefore concluded that the formation of the heme prosthetic group is closely integrated with the synthesis of the protein moiety. δ-Aminolevulinate synthase was detected in extracts of the organism. Its activity was correlated with cytochrome synthesis; it was reduced by high aeration and increased under low aeration. The synthase was inhibited by hemin at concentrations of 10 μmor higher. The observations are consistent with a central role for the heme prosthetic group in the regulation of cytochrome synthesis.

Respiration and mitochondria in Mucor genevensis, a facultatively anaerobic dimorphic mold, have been studied in aerobically and anaerobically grown cells and in anaerobically grown cells adapting to aerobic conditions. Respiration in hyphae continues at a high level during aerobic growth but drops rapidly on exhaustion of glucose. In anaerobically grown yeastlike cells, containing no recognizable aerobic cytochromes, a small cyanide-insensitive respiration occurs. Mitochondria with well defined cristae are visible in negative contrast after KMnO4fixation of stringently anaerobic cells containing low amounts of fatty acid of which 10% or less are unsaturated. On aeration of anaerobically grown cells, respiratory capacity and cytochromes develop rapidly, even in the presence of 10% glucose, indicating that glucose does not repress development of respiration. However, mycelium formation by adapting yeastlike cells is repressed by high glucose concentration. In adapting cells, apparent changes in mitochondrial ultrastructure appear to be more related to changes in fixation properties of cells than to changes in the structure of mitochondria.

CYTOPLASMICALLY inherited respiratory deficient mutants termed petites, were first described in baker's yeast over 20 years ago1. Since then in laboratory studies several species of yeast have been shown to give rise to respiratory deficient mutants2,3. However, although some respiratory deficient (obligatory fermentative) yeasts have been found in nature3, it is not clear whether these species are the result of cytoplasmic (petite) or chromosomal lesions4because no suitable genetic tests are available. Nevertheless it is known that petitemutants of both Saccharomyces cerevisiae5and Tornlopsis glabrata6have large deletions from their mitochrondrial DNA with each independent isolate having a unique circular DNA size profile which can be regarded as a ‘fingerprint’. By contrast chromosomal or segregational mutants have intact and functional mitochondrial DNA, as shown by genetic tests3,7,8. Therefore it seemed possible that the type of lesion in naturally occurring respiratory deficient species could be determined by circular DNA profile analysis. Using this method, we show here that three independently isolated strains of Candida slooffiiresemble petitemutants.