Changes in sexual agglutination ability during the formation of vegetative cells from spores in Saccharomyces cerevisiae

Summary Spores of heterothallic diploid cells of Saccharomyces cerevisiae had neither a nor agglutination substance in either cell wall or cytoplasmic fraction; they, however, showed selfagglutination not caused by sex-specific agglutination substances. Meanwhile, practically no sexual agglutination was detected during germination and outgrowth of the spores; it arose after emergence of the first buds and progressed with incubation time. Its ability increased gradualy until the first bud emergence and rapidly thereafter. a and agglutination substances were detected in both cell wall and cytoplasmic fractions of cells from an 8h-old spore culture. Only germinated spores with buds had the ability to produce and to respond to the a pheromone.     

Evidence for a functional membrane barrier in the transition zone between the flagellum and cell body of Chlamydomonas eugametos gametes

Evidence is presented which supports the concept of a functional membrane barrier in the transition zone at the base of each flagellum of Chlamydomonas eugametos gametes. This makes it unlikely that agglutination factors present on the surface of the cell body can diffuse or be transported to the flagellar membrane. The evidence is as follows: 1) The glycoprotein composition of the flagellar membrane is very different to that of the cell-body plasma membrane. 2) The flagella of gametes treated with cycloheximide, tunicamycin or , -dipyridyl become non-agglutinable but the source of agglutination factors on the cell body is not affected. 3) Even under natural conditions when the flagella are non-agglutinable, for example in vis-à-vis pairs or in appropriate cell strains that are non-agglutinable in the dark, the cell bodies maintain the normal complement of active agglutinins. 4) When flagella of living cells are labeled with antibodies bound to fluorescein, the label does not diffuse onto the cell-body surface. 5) When gametes fuse to form vis-à-vis pairs, the original mating-type-specific antigenicity of each cell body is slowly lost (probably due to the antigens diffusing over both cell bodies), while the specific antigenicity of the flagellar surface is maintained. Even when the flagella of vis-à-vis pairs are regenerated from cell bodies with mixed antigenicity, the antigenicity of the flagella remains matingtype-specific. 6) Evidence is presented for the existence of a pool of agglutination factors within the cell bodies but not on the outer surface of the cells.Abbreviations and symbols CHI cycloheximide - GTC guaniline thiocyanate - mt ⁺/mt ^- mating type plus or minus - PAS Periodic-acid-Schiff reagent - SDS sodium dodecyl sulphate     

Regulation of the production of the agglutination substances responsible for sexual agglutination in Saccharomyces cerevisiae: Changes associated with conjugation and temperature shift

Summary Isolated zygotes showed self-agglutination caused by the sex-specific glycoproteins, the agglutination substances responsible for sexual agglutination. The agglutination substances of both a and mating types were detected in the extracts obtained by the autoclave method from zygotes. Although the first diploid daughter cells from zygotes showed self-agglutinability, the self-agglutinability decreased gradually in the successive diploid daughter cells. The self-agglutination in diploid cells was also brought about by the complementary binding of the sex-specific agglutination substances of opposite mating types.The constitutive sexual agglutinability in a and cells was lost with concomitant loss of the agglutination substances in both cell wall and cytoplasmic fractions when cultured at a temperature higher than 35°C.The repression of the production of the agglutination substances was reversed by the opposite mating type pheromones even at the repressive temperature, 36°C, associated with the appearance of sexual agglutinability. The sex pheromones, a substance-I and substance-I, and the binding substance for substance-I were produced even at 36°C, repressive for the production of the agglutination substances.     

Mating reaction in Saccharomyces cerevisiae

Summary Haploid Saccharomyces yeasts showed sexual agglutination when a and type cells were mixed. Two types of a type strains were found; one constitutive and the other inducible concerning agglutinability. In type strains, no such differentiation was observed. Agglutination was inhibited by protease treatment. Secretion from type cells induced agglutinability in inducible a type cells. The activity of the secreted principle was heat-stable. The secretion is thought to induce de novo synthesis of proteinous sex-specific substances or to uncover preexisting sex substances.     

Secreted agglutinability-masking factors in Issatchenkia scutulata var. scutulata

The agglutinability-masking factors (AMFs) of a and mating types of Issatchenkia scutulata var. scutulata were prepared from culture fluids. AMFs masked the agglutinability of opposite mating-type cells sex-specifically, just like agglutination substances responsible for sexual cell agglutination. a AMF adsorbed to cells was eluted by incubating the cells at 60°C for 10 min. AMF was prepared directly from culture fluids of cells by DEAE-cellulose ion exchange chromatography. The active part of the AMFs is thought to be a peptidyl moiety because of the sensitivity to subtilisin. The pretreatment of cells with AMF of the opposite mating-type was shown to promote zygote formation. AMF slightly inhibited growth in a cells but not in cells, while a AMF did not show any growth-inhibitory effect on either a or x cells.     

The sexual agglutination substance is secreted through the yeast secretory pathway in Saccharomyces cerevisiae

Summary A Saccharomyces cerevisiae a strain carrying the secretory mutation sec1, sec7 or sec18 showed no sexual agglutination ability when treated with pheromone at the restrictive temperature 36° C, although the a agglutination substance had accumulated in the cytoplasm. These cells became sexually agglutinable, with a concomitant decrease in the agglutination substance in the cytoplasm, when the temperature was shifted from 36° C down to the permissive temperature 24° C after the addition of, cycloheximide. The a agglutination substance was barely detectable in sec53 cells (a) treated with pheromone at 36° C, indicating that the active a agglutination substance was formed after the export of its precursor into the endoplasmic reticulum. These results indicate that the a agglutination substance is exported through the yeast secretory pathway and that pheromone acts at the level of synthesis of the precursor molecule of the substance. An strain carrying sec1, sec7 or sec18 behaved just like an a strain carrying the sec gene in the induction of agglutination ability by the opposite mating type sex pheromone.     

Some properties ofSaccharomyces kluyveri

Four isolates of aSaccharomyces species which differed fromS. kluyveri by their ability to use cellobiose were analyzed genetically in relation to the latter species. Isolated single spores had low viability. Spore tetrads segregated mating types 2 2, with sexual agglutination occurring between complementary mating types. All single-spore isolates assimilated cellobiose indicating that these isolates were not naturally occurring hybrids betweenS. kluyveri and a cellobiose assimilatingSaccharomyces species.Two cell types were exhibited by single-spore cultures ofS. kluyveri, one granulated (G-type) and one vacuolated (g-type). G-type cultures formed fertile hybrids with complementary mating types of both G- and g-type cultures. Hybrids between two g-type cultures were sterile. They would, however, give fertile hybrids when mixed with G-type cultures.Sporulating hybrids betweenSaccharomyces sp. andS. kluyveri were produced. However the percentage spore germination was low. Single-spore cultures examined had cell types atypical of either parent. The ability to assimilate cellobiose was dominant and appeared to segregate with mating type and cell type.Weak mating reactions occurred when the (+) and (-) mating types ofSaccharomyces sp. were mixed with (a) and () mating types ofS. cerevisiae, respectively.The species ofSaccharomyces isolated from the Pacific Coast are designated as strains ofS. kluyveri.     

Mating-type differentiation in ascosporogenous yeasts on the basis of mating-type-specific substances responsible for sexual cell-cell recognition

Summary Sexual agglutination occurred only between cells of opposite mating types of the same species in all the Sacharomyces, Hansenula, Saccharomycodes, and Pichia yeasts tested. We succeeded in solubilizing the sex-specific glycoprotein, cell wall agglutination substance responsible for sexual agglutinability by briefly autoclaving these yeasts. The agglutination substances of all the above yeasts were univalent and sensitive to the enzyme pronase. The formation of complementary complexes was observed only between agglutination substances of opposite mating types of the same species. In general, the agglutination substance of one mating type was more resistant to heat treatment at 100°C in 3% acetic acid and more sensitive to 5% 2-mercaptoethanol treatment than the agglutination substance of the other mating type in these yeasts. On the basis of these results together with the pheromone response and production, we expect that almost all ascosporogenous yeasts can be classified into the two mating types corresponding to a and mating types in Saccharomyces cerevisiae, respectively.     

Binding of concanavalin A to intact cells and spheroplasts ofAnacystis nidulans

Native cells of the cyanobacterium (blue-green alga)Anacystis nidulans did not bind fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) as measured by fluorescent spectrophotometry. By contrast, spheroplasts ofA. nidulans underwent rapid and specific agglutination in the presence of ConA thus showing appreciable affinity towards the lectin. After treatment with 0.01–0.05% (wt/vol) cetyltrimethylammonium bromide (CTAB) intact cells also became liable to ConA binding, which was not accompanied by significant agglutination. Detergents, other than CTAB, were far less effective. Specific and nonspecific binding was discriminated, as usual, with the aid of methyl -d-mannoside. Conditions are described that allow specific binding of up to 7×10⁴ molecules of FITC-ConA per cell. The binding of ConA to pretreated cells ofA. nidulans was verified by freeze-etching electron microscopy using ferritin-ConA conjugate. Our results appear to be first to demonstrate lectin binding to a cyanobacterium.     

Mating reaction in Saccharomyces cerevisiae

A diffusible sex-specific substance called substance-I (S-I) was isolated from culture filtrate of type strains of the yeast Saccharomyces cerevisiae. The isolated S-I, an oligopeptide, induced sexual cell agglutinability in inducible a type strains and enhanced the agglutinability in constitutive a type strains. The induction of sexual agglutinability was detected in 30 min and reached maximum in 90 min, when 0.2 g/ml of S-I was added to inducible a type cells. The a type-specific factor responsible for sexual cell agglutination, called a type agglutination factor (aAF), was shown to be produced during the induction or the enhancement of agglutinability of a type cells by S-I. The aAF produced in response to S-I was not different in the susceptibility to proteolytic enzymes and disulfide-cleaving agents from those produced constitutively in the absence of S-I.     

Isolation,and biochemical and biological characterization of an a-mating-type-specific glycoprotein responsible for sexual agglutination from the cytoplasm of a-cells,in the yeast Saccharomyces cerevisiae

An a-mating-type-specific substance responsible for sexual agglutination was purified to 397-times in specific activity (units/mg protein) from the cytoplasm of a-mating type cells. The purified substance gave a single band stained with PAS reagent but not with both Coomassie brilliant blue and silver staining reagent by polyacrylamide gel electrophoresis in the presence of 8 M urea. However, incorporation of [³⁵S]methionine and Lowry reaction clearly indicate that the substance is a glycoprotein. The substance specifically masked sexual agglutinability of cells of the opposite mating type , indicating univalent action. The substance is a glycoprotein with a carbohydrate content of 90%, a pI of 4.5, and a molecular weight of 130,000. The substance was inactivated by 2-mercaptoethanol and proteolytic enzymes but not by glycolytic enzymes. The substance formed a complementary complex having no biological activity when mixed with -agglutination substance from the wall or cytoplasm of -cells in vitro.Non-common abbreviations PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

Sexual agglutination factors from the yeast pichia amethionina

Pichia amethionina is a heterothallic yeast isolated from necrotic cactus tissue. Haploid cells of opposite mating type, designated a and α, agglutinate strongly when mixed. The agglutination factors of the two cell types have been solubilized from the cell walls by β-glucanase digestion and then partially purified by affinity adsorption to the opposite cell type and by gel filtration. From α-cells was obtained a large, heat-stable glycoprotein with the ability to agglutinate a-cells. This α-agglutinin was inactivated by mercaptoethanol, probably because the recognition sites are linked to the glycoprotein core by disulfide bonds. Digestion of a-cells with β-glucanase released a large heat-labile glycoprotein that did not agglutinate α-cells but did inhibit agglutination of a-cells by α-agglutinin. Subtilisin digestion of this a-factor released a carbohydrate-free protein of 27,000 daltons that retained the biological activity of the factor. These agglutination factors are sex- and species-specific and are not found on the surface of heterozygous diploid cells.     

The release of sex-specific substances responsible for sexual agglutination from haploid cells of Saccharomyces cerevisiae

Cell surface substances responsible for sexual cell agglutination were successfully released in a large quantity from heterothallic haploid cells of Saccharomyces cerevisiae by a newly established autoclaving method. The conditions for this releasing phenomenon were examined. The sexual agglutination substances were solubilized most efficiently when the cells, suspended in a 30 mM Tris-HCl, pH 7.0, 5 mM EDTA solution, were autoclaved at a pressure of 1 kg/cm² at 120 °C for 3 min. The substances were specifically adsorbed by the cell surface of the opposite mating type, resulting in the masking of agglutinability of the cells of the opposite mating type. The substances were not released from the surface of cells which lacked sexual cell agglutination. The evidence suggesting the formation of a molecular complex between a- and α-agglutination substances in vitro is also presented. The above procedure is applicable to the solubilization of surfage agglutination substances from various strains of S. cerevisiae.     

Sexual cell agglutination in relation to the formation of zygotes in Saccharomyces cerevisiae

The ratio of a to cells in the sexual cell aggregates was consistentlyabout one regardless of the ratio of a to cells at the initialmixing. Conjugating cells seemed to be formed exclusively inthe aggregates during mixed culture of a and cells. Large cellswith buds (L cells) and small cells without buds (S celb) wereseparated from a logarithmic culture by sucrose density gradientcentrifugation. L Cells showed higher sexual agglutinabilitythan S cells in a mating type, but such difference was not detectedin a mating type. The same tendency was observed in cells dividingsynchronously. Based on the above results, the biological significanceof sexual agglutination in the mating reaction is discussed. ¹ Present address: Department of Physiology, Japan Women's University,Bunkyo-ku, Tokyo 112, Japan. (Received September 8, 1978; )     

Growth and metabolic flexibility in groundwater bacteria

Groundwater bacteria isolated from an oligotrophic-saturated soil showed a mixed strategy of economized metabolism and migration when grown in a continuous-flow column system simulating poor or nutrient-amended growth conditions. The cells were generally <0.5m in diameter in pure groundwater, but doubled in size when the concentration of dissolved organic carbon and phosphate in groundwater was increased 10-fold. The biomass, estimated from analysis of muramic acid (MuAc) in cell wall peptidoglucans, increased at the same time by a factor of 5 when the solid support in the columns was gravel and by a factor of 10 when it was glass beads. Bacteria in pure groundwater stored 10 times more of the energy-rich polysaccharide, poly--hydroxybutyric acid (PHB), than bacteria in enriched groundwater, and those cells that were attached to the gravel stored 10 times as much as cells in the interstitial pore water. Once phosphate was added to groundwater, stored PHB was metabolized. The proportion of free-living to attached bacteria was 2 to 10 times higher in enriched compared with pure groundwater indicating a mass transport of cells as the carrying capacity of their habitat rose.     

Genetics of a-agglutunin function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p). Although functional a-agglutinin is expressed only by a cells, previous results indicated that AGA1 RNA is expressed in both a and cells after pheromone induction. Expression of the Aga2p adhesion subunit in a cells allowed a-agglutinability, indicating that a cells express the a-agglutinin anchorage subunit, although no role for Aga1p in cells has been identified. Most of the a-specific agglutination-defective mutants isolated previously were defective in AGA1; a single mutant (La199) was a candidate for an aga2 mutant. Expression of AGA2 under PGK control allowed secretion of active Aga2p from control strains but did not complement the La199 agglutination defect or allow secretion of Aga2p from La 199, suggesting that the La199 mutation might identify a new gene required for a-agglutinin function. However, the La199 agglutination defect showed tight linkage to aga2::URA3 and did not complement aga2::URA3 in a/a diploids. The aga2 gene cloned from La199 was nonfunctional and contained an ochre mutation. The inability of pPGK-AGA2 to express functional Aga2p in La199 was shown to result from an additional mutation(s) that reduces expression of plasmid-borne genes. AGA2 was mapped to the left arm of chromosome VII approximately 28 cM from the centromere.     

Auxin increases the hydraulic conductivity of auxin-sensitive hypocotyl tissue

The ability of water to enter the cells of growing hypocotyl tissue was determined in etiolated soybean (Glycine max (L.) Merr.) seedlings. Water uptake was restricted to that for cell enlargement, and the seedlings were kept intact insofar as possible. Tissue water potentials ( _w) were measured at thermodynamic equilibrium with an isopiestic thermocouple psychrometer. _wwas below the water potential of the environment by as much as 3.1 bars when the tissue was enlarging rapidly. However, _w was similar to the water potential of the environment when cell enlargement was not occurring. The low _w in enlarging tissue indicates that there was a low conductivity for water entering the cells.The ability of water to enter the enlarging cells was defined as the apparent hydraulic conductivity of the tissue (Lp). Despite the low Lp of growing cells, Lp decreased further as cell enlargement decreased when intact hypocotyl tissue was deprived of endogenous auxin (indole-3-acetic acid) by removal of the hypocotyl hook. Cell enlargement resumed and Lp increased when auxin was resupplied exogenously. The auxin-induced increase in Lp was correlated with the magnitude of the growth enhancement caused by auxin, and it was observed during the earliest phase of the growth response to auxin. The increase in Lp appeared to be caused by an increase in the hydraulic conductivity of the cell protoplasm, since other factors contributing to Lp remained constant. The rapidity of the response is consistent with a cellular site of action at the plasmalemma, although other sites are not precluded.Because the experiments involved only short times, auxin-induced changes in cell enlargement could not be attributed to changes in cell osmotic potentials. Neither could they be attributed to changes in turgor, which increased when the rate of enlargement decreased. Rather, auxin appeared to act by altering the extensibility of the cell walls and by simultaneously altering the ability of water to enter the growing cells under a given water potential gradient. The hydraulic conductivity and extensibility of the cell walls appeared to contribute about equally to the control of the growth rate of the hypocotyls.     

The attachment of bacterial cells to surfaces under anaerobic conditions

The cell fixation of Pseudomonas aeruginosa, Citrobacter amalonaticus, and Methanosarcina barkeri to lipid-free glass slides was investigated under anaerobic conditions using cell number, protein content, and ATP level as the biomass parameters. Energy substrates stimulated the attachment of P. aeruginosa and C. amalonaticus significantly as compared to the fixation behaviour in basal medium without substrates. The fixation exhibited charateristic kinetics of attachment and detachment of the cells. Cells of M. barkeri obtained from pure cultures did not adhere at all. In mixed cultures with C. amalonaticus, significant fixation of M. barkeri cells was observed. Light micrographs gave rise to the assumption that M. barkeri was incorporated into already existing biofilms. This mechanism is supposed to be important for retaining methanogenic biomass in anaerobic biofilms. The results could help to reduce the start-up periods of biofilm reactors and to enhance methanogenic activities in this environment. This assumption was supported by experiments performed with methanogenic mixed cultures fixed to ceramic particles in biofilm reactors. Correspondence to: M. Meier-Schneiders