Hisao Miyata

Lysis of growing fissin-yeast cells induced by aculeacin A, a new antifungal antibiotic

Cells of Schizosaccharomyces pombe grown in the presence of aculeacin A, a peptide antibiotic, were lysed resulting the death of cells. Under high osmolarity, the cellular lysis induced by aculeacin A was considerably reduced. The use of synchronous-culture systems distinguished cell elongation from cell division revealed that the sites of aculeacin A-induced lysis on the fission yeast were the end(s) and the cell plate region, corresponded to the regions of the cell wall synthesis. Aculeacin A-resistant survivors exhibited morphological alterations which were swollen at one or both ends of the cell and appeared drumstick or dumbbel like; the wall of the bulge region was observed to be stained with a fluorescent brightener, as well as that of the cell plate region. These effects of aculeacin A are discussed as compared with effects of 2-deoxy-D-glucose.

Sexual co-flocculation by heterothallic cells of the fission yeast Schizosaccharomyces pombe modulated by medium constituents.

Novel simple synthetic media for inducing sexual co-flocculation in a short time after mixing heterothallic fission-yeast (Schizosaccharomyces pombe) cells of h- and h+ were devised; The most effective of these, mannose synthetic medium (MSM), contains 0.4% mannose as a carbon source in addition to galactose, KH2PO4 (pH4.0) and 4 vitamins. The addition of galactose to the medium suppressed the asexual self-flocculation but rather promoted the sexual co-flocculation. By transferring and mixing h- and h+ cells grown in malt-extract broth plus galactose into MSM, these heterothallic strains were revealed to be sexually ready through a long period of the log to stationary phases. Furthermore, a variety of C sources and NH4Cl at various concentrations in various media were examined for their effects upon sexual co-flocculation, conjugation and sporulation; it was found that the sugar concentration strictly affected the progress of the sequence of sexual reproduction at 26°C but not 30°C and that sexual co-flocculation of the heterothallic strains was induced only under lower concentrations of C and N source than that for the homothallic one.

Asymmetric Location of the Septum in Morphologically Altered Cells of the Fission Yeast Schizosaccharomyces pombe

Summary: Cells of the fission yeast Schizosaccharomyces pombe, normally sausage-shaped, changed to a round-bottomed flask (RBF)-like morphology during growth in the presence of aculeacin A (Acu), an antifungal antibiotic. The volume of RBF-like cells was comparable to that of the control cells. After being transferred to normal conditions (without Acu at 25°C), the RBF-like cells continued to grow at the cylindrical and/or spherical end(s) and then the septum at the subsequent division of the cells was formed without exception at the boundary plane between the spheroidal and the cylindrical region; it is at this boundary that the nucleus was located before mitosis. Hence the RBF-like cell divided into a spheroidal and a cylindrical sib at the first cell division. At the end of the second cell cycle, the spheroidal and the cylindrical progeny divided into two spheroidal and two cylindrical sibs respectively. The values of the mean length (long/short) and volume (big/small) ratios of paired sibs were larger in order of (a) cylindrical normal, with both mean ratios 1·06; (b) cylindrical control; (c) cylindrical progeny of RBF-like cell; (d) spheroidal progeny of RBF-like cell; and (e) RBF-like cell, whose mean length ratio was 1·25 but whose mean volume ratio was 1·94. That is, the more the morphology deviated from the cylindrical form, the greater was the degree of asymmetry. There was no rule relating the biases to the growth pole in these asymmetries.

Sibling differences in cell death of the fission yeast, Schizosaccharomyces pombe, exposed to stress conditions.

Cell division of the sausage-shaped fission yeast, Schizosaccharomyces pombe , is considered to be a typical binary fission (Mitchison 1970; Johnson et al. 1979; Miyata et al. 1986b). The last stages of fission are often very slow, leaving two sibs attached by a thread of old cylindrical wall to be a V-form for a while (Mitchison 1970). These we call ‘V-pairs’. The resultant siblings are suitably considered to be ‘sisters’ rather than ‘mother and daughter’, because just after division they have almost the same morphology and size. The most obvious morphological differences between sibs are (a) their numbers of fission scars (Johnson & Lu 1975; Calleja et al. 1980) that are usually proximal to cylindrical regions having increased diameter (Johnson & Lu 1975) and cell wall thickness (Streiblovà et al. 1966; Johnson et al. 1995), and (b) the related differences between sibs lengths after division (Johnson et al. 1979; Miyata et al. 1986b). Microbiologists generally deal with fission yeast cells only as members of a population, hence there are no reports about differences between V-pair sibs. However, we have noted that one of the V-pair sibs often died while the other survived after the culture was transferred to extreme, but sub-lethal conditions. We ask therefore if one-sib death tends to be specific for either of the V-pair sibs. If so, how do the sibs ‘ages’ bear upon that ‘selective death’? In this work, the responses to stress conditions that take place within one cell cycle, that is, ‘early, primary, or immediate responses’ according to Ruis (1997), were analyzed in relation to differences of scar numbers and extension growth between sibs in V-pairs where selective death has occurred (see below). Except where noted, 17 hr-grown (late-log phase) yeasts in Edinburgh Minimum Medium No. 2 (EMM 2, pH 4.5; Mitchison 1970) at 32 ◦C were exchanged to fresh EMM2 at a density of 5× 106 or 1 × 107cells/ml and then incubated at 32 ◦C. Three extreme conditions were used as follows: the temperature (temp.) up shift was performed by two steps, i.e., cells were incubated at 37 ◦C for 30 min and then the temperature was shifted up to 41 ◦C; pH shift-up was by adjustment to pH 7.0 with 0.05 M K-phosphate buffer; acridine orange (AO) addition was to 200 μg/ml. All cultures were shaken reciprocally. Scar number on a cell stained with Calcofluor White New(CFW) and dead cells was determined by using an Olympus BHS-RFK epifluorescence microscope (Tokyo, Japan) with phase-contrast equipment. The sib with damaged or disorganized cytoplasm (phase-light) or was freely permeable to AO was defined as dead (Figure 1). Sensitivity exhibited as cell death of five strains of Schizosaccharomyces pombe IFO 0365 (Miyata and Miyata 1978), 972 h −, 975 h+, 968 h90, and NCYC 132 was observed within 2.0 h of transfer to one of the extreme conditions (Table 1). Strains IFO 0365 and 972 h−exhibited similar extents of sensitivity to the three kinds of stress. On the other hand, 975 h +, 968 h90 and NCYC 132 were about as sensitive to AO as the former strains but had little or no sensitivity to pH or temp. shift-up. Accordingly, IFO 0365 and 972 h −

Establishment of Septum Orientation in a Morphologically Altered Fission Yeast, Schizosaccharomyces pombe

SUMMARY: Among the spheroidal fission-yeast (Schizosaccharomyces pombe) cells resulting from aculeacin A treatment were found cells whose putative growth axis and polarity differed from those of their progenitor, that is they were changed in the orientation of their septum. The ratio of cell length (measured perpendicular to the septum plane) to septum diameter of these cells equalled or exceeded unity without exception, whether the septum orientation changed or not and whether the cellular shape was spherical, spheroidal or cylindrical. From these results we conclude that the septum is always oriented perpendicular to the plane including the longest axis of the cell even if the morphology is irregular or the new septum has become perpendicular to the previous septum. There is no cellular region forbidden to new septa.