Physiological aspects of conjugation in fission yeast

Summary Conjugation was studied in Schizosaccharomyces pombe using liquid media. Nitrogen, which was growth-limiting in a synthetic medium, had to be consumed completely before conjugation could start. Conjugation was preceded by sexual agglutination. Agglutinability was not constitutive in heterothallic strains. It only developed when cells of h ⁺ and h ^- mating type were grown in mixed culture for at least 2.5 hr before the start of conjugation.     

Induction of trehalase activity on a nitrogen-free medium: a sporulation-specific event in the fission yeast, Schizosaccharomyces pombe

Summary Kinetic experiments with synchronously sporulating cultures of a homothallic h⁹⁰ strain of Schizosaccharomyces pombe showed that trehalase activity abruptly increased in the late sporulation process, coinciding with the appearance of visible spores. Trehalase activity was absent in vegetative cells. A set of strains different in genetic constitution at the mating type loci was tested for induction of trehalase on nitrogen-free sporulation medium. The appearance of trehalase activity on the sporulation medium was observed only in sporulating cultures; cultures of homothallic strains (h⁹⁰) and diploid strains heterozygous for mating type (h⁺/h^–), and mixed cultures of heterothallic h⁺ and h^– strains. Trehalase activity was not induced in nonsporogenic strains: heterothallic haploid strains (h⁺ and h^–), diploid strains homozygous for mating type (h⁺/h⁺ and h^–/h^–) and the homothallic strain harboring the mutation in the mat2 gene, which was unable to undergo the first meiotic division. Trehalose accumulation on the sporulation medium was observed solely in the sporulating cultures. These results led us to conclude that the induction of trehalase activity as well as the accumulation of trehalose in the medium lacking nitrogen sources was a sporulation-specific event under the control of the mating type genes.     

Induction of sexual co-flocculation of heterothallic fission yeast (Schizosaccharomyces pombe) cells by mating pheromones

Heterothallic fission yeast (Schizosaccharomyces pombe) cells preincubated with sex pheromone, P- or M-factor of the obverse mating-type cells, in mannose synthetic medium (MSM) results in remarkably increased sexual co-flocculation with obverse mating-type cells almost without time lag, i.e., within 10 min. By contrast, comparable flocculation requires over 1 h if untreated control cells are mixed with obverse mating-type cells. The agglutinin of P cells is more inducible than that of M cells. These pheromonal inductions of sexual co-flocculation are inhibited by the addition of cycloheximide or tunicamycin during preincubation but not by chloramphenicol or hydroxyurea. These results demonstrate that, in addition to (a) the repression of cell division (G1 arrest) and (b) the activation of cell wall autolytic processes (mating-specific elongation of cells: formation of their conjugation tubes), mating pheromones of fission yeast have another important role; (c) to induce sexual co-flocculation (agglutinability). Using our experimental system of preincubation with sexual pheromones, we show that M-agglutinin is heat-stable and its induction is inhibited by tunicamycin, but that P-agglutinin is heat-labile and its induction is only partially inhibited by tunicamycin.     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

The occurrence of killer,sensitive, and neutral yeasts in Brazilian Riesling Italico grape must and the effect of neutral strains on killing behaviour

 The occurrence of killer toxins amongst yeasts in Brazilian Riesling Italico grape must was investigated by using the sensitive strain EMBRAPA-26B as a reference strain at 18°C and 28°C. From a total of 85 previously isolated yeasts, 21 strains showed ability to kill the sensitive strain on unbuffered grape must/agar (MA-MB) and 0.1 M citrate/phosphate-buffered yeast extract/peptone/dextrose/agar (YEPD-MB) media both supplemented with 30 mg/l methylene blue. The killer activity of only four yeasts depended on the incubation temperature rather than the medium used. At 28°C, the strains 11B and 53B were not able to show killer action. On the other hand, strains 49B and 84B did not kill the sensitive yeast at 18°C. The killer strain EMBRAPA-91B and a commercial wine killer yeast K-1 were employed to examine the sensitivity of the isolated yeasts on YEPD-MB and MA-MB at 18°C. The sensitivity and neutral characteristics of yeasts were shown to be dependent on the medium and the killer strain. Interactions, including K^- R^-, K^- R⁺ and K⁺ R⁺ strains, simultaneously, have revealed that some K^-R⁺ strains appear to protect the K^- R^- strain against the killer toxin. Sensitive dead cells, although to a less extent, also exhibited similar protection. Kinetic studies have shown that the maximum specific growth rates were higher for the 20B YEPD-MB-sensitive strain (μ_max=0.517 h^-1) than for both the 91B (μ_max=0.428 h^-1) and K-1 (μ_max= 0.466 h^-1) killer strains. The protective capacity of neutral or sensitive cells that contaminate a fermentation, as well as the higher maximum specific growth rate of sensitive yeasts, besides other factors, may preclude the dominance of a killer strain. This protective capacity may also reduce the risk of a sensitive inoculum being killed by wild-type killer yeasts in open non-sterile fermentation. Received: 3 November 1995/Received revision: 11 March 1996/Accepted: 15 April 1996     

REPRODUCTIVE ISOLATION BY SEX PHEROMONES IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (ZYGNEMATALES,CHAROPHYCEAE)1

The Closterium peracerosum–strigosum–littorale (C. psl.) complex consists of unicellular algae and is known to be composed of several reproductively isolated mating groups of heterothallic strains. Group I‐E is completely isolated from mating groups II‐A and II‐B, groups II‐A and II‐B are partially isolated from each other, and only mating‐type plus (mt⁺) cells of group II‐A and mating‐type minus (mt^?) cells of group II‐B form zygotes. Based on the alignment of 1506 group I introns, significant phylogenetic relationships were observed among mating groups II‐A and II‐B, while mating group I‐E was distant from groups II‐A and II‐B. Sexual cell division in both mating‐type cells of group II‐A was stimulated in conditioned media in which cells of group II‐B had been cultured. When mt^? cells of group II‐B were stimulated in conditioned medium derived from group II‐A, mt⁺ cells of group II‐B did not respond to the conditioned medium. Conditioned media derived from group I‐E did not exhibit sexual cell division (SCD)–inducing activity against any strain except those within its own group. From the alignment of deduced amino acid sequences from orthologous protoplast‐release‐inducing protein (PR‐IP) Inducer genes, we detected a significant similarity among groups II‐A and II‐B, and mating group I‐E had low similarity to other mating groups. The existing degree of reproductive isolation can be partially explained by differences in molecular structures and physiological activities of sex pheromones of these heterothallic mating groups.     

Structural analysis of galactomannans by NMR spectroscopy

The structure of naturally occurring galactomannans was characterized by high resolution NMR spectroscopy involving two-dimensional (2D) NMR measurements of the field gradient DQF-COSY, HMQC, HMBC, and ROESY experiments. Four galactomannans with different proportions of galactose (G) and mannose (M), from fenugreek gum (FG), guar gum (GG), tara gum (TG), and locust bean gum (LG), were investigated. Because these galactomannans had very high molecular weights, hydrolysis by dilute H₂SO₄ was carried out to give the corresponding low molecular weight galactomannans, the structural identities of which were established by comparison of the specific rotations, shape of the GPC profiles, and NMR spectra with those of higher molecular weight galactomannans. The correlation signals GH1-GC4, -GC5, and -MC6 in HMBC and GH1-GH6 in ROESY spectra of FG showed that more than two galactopyranose units with the 1 → 4 linkage were connected at C6 of the mannopyranose main chain. The coupling constant (J_H1,2) of galactose was 3.4 Hz, indicating that galactose has an α-linkage. The main chain mannose was found to connect through the 1 → 4 linkage, because of the appearance of the correlation signals MH1-MC4, and MC1-MH4 in the HMBC spectrum due to the long-range correlation signals between two neighboring mannopyranose residues through the M4-O-M1 bond. Although the main chain mannose J_H1,2 was not observed, probably because of the high molecular weight, the specific rotation of LG with a higher proportion of mannose was low, [α]_D²⁵ = +10.8°, compared with that of FG with a lower proportion of mannose, [α]_D²⁵ = +90.5°, suggesting that the mannose in the main chain had a α-linkage. These results suggest that the galactomannans comprise a (1 → 4)-β-mannopyranosidic main chain connected with more than two (1 → 4)-α-galactopyranosidic side chains, in addition to the single galactopyranose side chain, at C6 of the mannopyranose main chain.     

Occurrence of O-methylated sugars in surface glycoconjugates in Chlamydomonas eugametos

Previously, we have shown that the monomeric-sugar composition of cell-surface-associated glycoconjugates of two strains of Chlamydomonas eugametos, of different mating type, differs strikingly (Gerwig et al. 1984, Carbohydr. Res. 127, 245–251). Besides the common occurrence of various pentoses and hexoses, the glycoconjugates of one strain contain 4-O-methyl xylose, a 2-O-methyl pentose (probably 2-O-methyl arabinose) and 3-O-methyl galactose, whereas those of the other strain contain 6-O-methyl mannose and 3-O-methyl glucose. In order to investigate whether these differences are relevant to the mating process of this organism, the sugar composition of the sexual progeny of these strains was analyzed. The ability to produce 4-O-methyl xylose, 2-O-methyl pentose and 3-O-methyl galactose on the one hand, and the ability to produce 6-O-methyl mannose and 3-O-methyl glucose on the other hand, appear to be genetically linked. However, the ability to produce either set of O-methyl sugars was inherited independently of mating type. O-Methylated sugars do not occur in the cell wall of C. eugametos, or in the cell-free medium, but only in surface-membrane-associated glycoconjugates, extractable with salt or detergent solutions.Abbreviation mt ^+/- mating-type plus or minus     

Zygospore formation between homothallic and heterothallic strains of Closterium

Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt⁻) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt⁺ cells, compared with that in the homothallic strain alone and in a coculture with mt⁻ cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt⁺ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt⁻-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.     

Comparison of Extracellular Polysaccharide Composition, Rhizobitoxine Production, and Hydrogenase Phenotype among Various Strains of Bradyrhizobium japonicum

A survey of 51 strains of Bradyrhizobium japonicum was performedwith respect to the composition of extracellular polysaccharide(EPS), the production of rhizobitoxine and the hydrogenase phenotype.A good correlation was found among these three different characteristics.Thirty-six strains producing an EPS composed of glucose, mannose,galactose, 4-O-methyl galactose and galacturonic acid did notsynthesize rhizobitoxine, whereas 14 strains producing an EPScomposed of rhamnose and 4-O-methyl glucuronic acid were allfound to synthesize rhizobitoxine. Hydrogen-uptake positive(Hup⁺) strains were confined exclusively to the former groupof strains which produced an EPS composed of glucose, mannose,galactose, 4-O-methyl galactose and galacturonic acid. Theseresults suggest that the phenotype with respect to rhizobitoxineproduction and hydrogen uptake is involved in the phylogenyof Bradyrhizobium japonicum as well as in the productivity ofnodulated soybeans. (Received March 24, 1989; Accepted June 16, 1989)     

Dissection of conjugants and mating type plus and minus cells in selfing clones of the isogamous green alga Closterium ehrenbergii

In the sexual reproduction of the green alga Closterium ehrenbergii, two sexually competent cells that are morphologically indistinguishable from the vegetative cells first come close to each other to form a sexually interacting pair. Each then divides into two gametangial cells. Isogamous conjugation occurs between nonsister gametangial cells of the two resulting pairs. With unusual selfing clones derived from a certain cross of heterothallic strains, we dissected apart a pair of gametangial cells that had already been united together by a delicate transparent tube, into which each gametangial cell was going to develop its conjugation papilla. In spite of such a degree of differentiation, when each was cultured in fresh medium, individual gametangial cells could dedifferentiate into vegetative cells and form subclones. By crossing such subclones with standard stable heterothallic mating-type strains, we show that each selfing clone of this alga actually produces both stable mt ⁺ and stable mt ^- cells, in addition to unstable mt ^- cells with selfing potency, during its mitotic vegetative growth. Although the selfing in C. ehrenbergii studied here differs in certain points from true homothallism, the results of the present study provide insight into how homothallism might have evolved from heterothallism.     

Isolation and preliminary characterization of mutants of the cyanobacterium Nostoc muscorum resistant to growth inhibition by methylamine

Summary The wild-type heterocystous and nitrogen-fixing (Het ⁺ Nif ⁺) N. muscorum and its non-heterocystous non-nitrogen-fixing (Het ^- Nif ^-) mutant strain both fail to grow in different inorganic nitrogen media containing 1 mM methylamine hydrochloride (MA). Mutants of the Het ⁺ Nif ⁺ and Het ^- Nif ^- parents resistant to growth inhibition by 5 mM MA and thus designated as MA ^R strains were isolated with a frequency of 2.5(±2.4)×10⁶. A MA ^R strains of the Het ⁺ Nif ⁺ and a MA ^R strain of the Het ^- Nif ^- parent were characterized for growth, heterocyst formation and acetylene reducing activity in the presence and absence of methylamine in N₂ medium. The Het ⁺ Nif ⁺ MA ^R strain grows better in MA containing than in MA-free N₂ medium, and all cultures grown with MA are found to lack both acetylene reducing activity and heterocyst. The Het ^- Nif ^- MA ^R strain shows good growth in MA-containing N₂ medium but no growth in MA-free N₂ medium. Furthermore, both the Het ⁺ Nif ⁺ MA ^R and Het ^- Nif ^- MA ^R strains show better growth in the presence than in the absence of MA in NO ₃ ^- and NH ₄ ⁺ media. These results appear to suggest that the MA ^R phenotype in N. muscorum is due to the metabolic utilization of the ammonium analog as a nitrogen source.     

Effect of vesicular-arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria on growth and nutrition of soybean in a neutral-calcareous soil amended with32P-45Ca-tricalcium phosphate

Summary The optimum conditions for growth ofFrankia sp. HFPCcI3 isolated fromCasuarina cunninghamiana, were studied in batch culture using defined media. Maximum growth (doubling time was 24 h)_was achieved at 33°C and at pH 6.3 with pyruvate and NH ₄ ⁺ as the sole C and N sources, respectively. Removal of NH ₄ ⁺ from the culture medium resulted in vesicle differentiation which was paralleled by induction of acetylene reduction activity. Growth on atmospheric N₂ was optimal with combined pyruvate and glucose as the carbon sources and displayed a doubling time of about 48 h. Comparisons in growth and N₂-fixing activity ofFrankia strains grown in a variety of cultural conditions demonstrate the range of behavior among the strains.     

Temperature dependence of inorganic nitrogen uptake and assimilation in Antarctic sea-ice microalgae

Summary The influence of temperature on NO ₃ ^- and NH ₄ ⁺ uptake, and the activity of the assimilatory enzyme NO ₃ ^- reductase (NR) was compared to inorganic C uptake (photosynthesis) in natural assemblages of Antarctic sea-ice microalgae. NO ₃ ^- and NH ₄ ⁺ uptake reached a maximum between 0.5°–2.0°C and 2.0°–3.0°C, respectively, which was close to that for photosynthesis (2.5°–3.0°C). NR showed a distinctly higher temperature maximum (10.0°–12.0°C) and a lower Q₁₀ value than inorganic N and C transport. Our data imply that, owing to differential temperature characteristics between N transport and N assimilation at in situ temperature (-1.9°C), the incorporation of extracellular NO ₃ ^- into cellular macromolecules, may be limited by transport of NO ₃ ^- into the cell rather than the intracellular reduction of NO ₃ ^- to NH ₄ ⁺ . Despite differences in temperature maxima between N transport and N assimilation, the overall low temperature maxima of inorganic N metabolism characterizes Antarctic sea-ice microalgae as psychrophilic. Our study is the first to examine the temperature dependence of inorganic N uptake and assimilation in sea-ice microbial communities.     

Glycoproteins in bacterial membranes. In vivo labeling of the sugar portion of energy-transducing ATPase and a low-molecular-weight fraction fromMicrococcus lysodeikticus membranes

The energy-transducing ATPase and a low-molecular-weight fraction ofMicrococcus lysodeikticus membranes incorporated¹⁴C label fromd-[U-¹⁴C]glucose fed to the bacteria in synthetic medium. The specific radioactivity of the sugar portion of the ATPase and low-molecular-weight fraction was, respectively, 2.65 and 2.88 times that of their amino acids. Glucose and mannose in approximately equimolar amounts were identified as the main sugars of the glycoprotein ATPase, thus confirming previous structural studies. Glucose, galactose, and mannose (1:1:2) were identified as the main sugars of the low-molecular-weight glycopeptides. These results confirm and extend the notion that glycoprotein are constituents of prokaryotic membranes.     

The effect of the source of inorganic nitrogen on growth and enzymes of nitrogen assimilation in soybean and wheat cells in suspension cultures

Summary Soybean (Glycine max L. cv. Mandarin) and wheat (Triticum monococcum L.) cells were grown in media with NO₃ ^- plus NH₄ ⁺ (B₅) and NO₃ ^- without NH₄ ⁺ (B₅-NH₄) as nitrogen sources. Changes in pH, [NO₃ ^-] and [NH₄ ⁺] in media, and dry weight, protein content, nitrate reductase (NR) and glutamate dehydrogenase (GDH) in the cells were followed for about 170 h. With both NH₄ ⁺ and NO₃ ^- in the medium, NH₄ ⁺ was utilized very quickly. Soybean cells grew poorly in the absence of NH₄ ⁺ while wheat cells grew equally well on media with or without NH₄ ⁺. When soybean cells were grown in medium with NO₃ ^- plus NH₄ ⁺, dry weight and NR activity remained relatively low for several hours after which both increased rapidly. This coincided with the time NH₄ ⁺ was depleted from the medium. In the absence of NH₄ ⁺, soybean cell growth and NR activity remained low. NR activity in wheat cells, and GDH activity in soybean and wheat cells, did not vary significantly in the presence or absence of NH₄ ⁺.This work was supported by a grant in aid of research from the National Research Council of Canada to one of us (J. K.). NRCC No. 12521.     

Schistosoma mansoni: glycosyl transferase activity and the carbohydrate composition of the tegument.

Homogenates of adult Schistosoma mansoni contain enzymes which transferred [¹⁴C]mannose, [¹⁴C]glucose, and [¹⁴C]galactose from GDP-[U-¹⁴C]mannose, UDP-[U-¹⁴C]glucose, and UDP-[U-¹⁴C]galactose respectively to a lipid acceptor; in comparison, free [¹⁴C]mannose, GDP-[U-¹⁴C]fucose, and UDP-[U-¹⁴C]acetyl-glucosamine were poorly transferred. The lipid acceptor is believed to be an intermediate in the glycosylation of the worm's glycoproteins and in the biosynthesis of oligosaccharides and glycolipids. The tegument of adult worms was isolated by the freeze-thaw procedure and sugars associated with macromolecules in this fraction were analyzed; the major monosaccharide components were glucose, galactose, and mannose. These results suggest that the mechanism of glycosylation of the adult schistosome's tegumental macromolecules may occur through the glycosyl transferase system. The schistosome mannosyl transferase (EC 2.4.1), which is membrane bound was solubilized with 0.1% Triton X-100 without loss of activity; after density gradient centrifugation there was a peak of enzymic activity in a region of density 1.08, which could not be associated with any particular organelle.     

Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K−Cl cotransport

Pathways of K⁺ movement across the erythrocyte membrane of frog Rana temporaria were studied using ⁸⁶Rb as a tracer. The K⁺ influx was significantly blocked by 0.1 mmol·l^-1 ouabain (by 30%) and 1 mmol·l^-1 furosemide (by 56%) in the red cells incubated in saline at physiological K⁺ concentration (2.7 mmol·l^-1). Ouabain and furosemide had an additive effect on K⁺ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K⁺ influx saturated as f(K⁺)_e with apparent K _m values for external K _e ⁺ concentration of 0.96±0.11 and 4.6±0.5 mmol·l^-1 and V _max of 0.89±0.04 and 2.8±0.4 mmol·l cells^-1·h^-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of K _e ⁺ medium concentration. Total K⁺ influx was significantly reduced when frog erythrocytes were incubated in NO _- ³ medium. Furosemide did not affect K⁺ transport in frog red cells in NO ₃ ^- media. At the same K _e ⁺ concentration the ouabain-furosemide-insensitive K⁺ influx in Cl^- medium was significantly greater than that in NO _- ³ medium. We found no inhibitory effect of 1 mmol·l^-1 furosemide on Na⁺ influx in frog red cells in Cl^- medium. K⁺ loss from the frog erythrocytes in a K⁺-free medium was significantly reduced (mean 58%) after replacement of Cl^- with NO _- ³ . Furosemide (0.5 mmol·l^-1) did not produce any significant reduction in the K⁺ loss in both media. The Cl^--dependent component of K⁺ loss from frog red cells was 5.7±1.2 mmol·l^-1·h^-1. These results indicate that about two-thirds of the total K⁺ influx in frog erythrocytes is mediated by a K–Cl cotransport which is only partially blocked by furosemide.Abbreviations DMSO dimethyl sulphoxide - K _e ⁺ external concentration of K⁺ - K _m apparent Michaelis constant for external - K⁺ K _e ⁺ at V _max/2 - RBC red blood cell(s) - V _max maximal velocity of the unidirectional K⁺ influx - TRIS tris(hydroxymethyl)aminomethane     

Cell Fusion Promoting Factor Common to Homothallic and Heterothallic Mating Systems in Dictyostelium discoideum1

Cellular slime mould Dictyostelium discoideum propagates as single haploid cells and under certain environmental conditions enters into a sexual cycle called macrocyst formation. There are homothallic and heterothallic strains reported, the former being able to form macrocysts in clonal cell populations while the latter to do so only in the presence of opposite mating-type strains. Molecular basis for differential mating systems is an intersting subject totally unknown yet. In the present study, sexual cell interactions in AC4, a homothallic strain of D. discoideum, was studied in comparison with the heterothallic mating system. The conditoned medium of AC4 cells was found to promote the sexual cell fusion among themselves. In addition, it also enhanced the cell fusion between heterothallic strains. Furthermore, the conditioned medium obtained from the mated culture of heterothallic strains reported to induce the sexual cell fusion in the heterothallic strains (Saga and Yanagisawa, 1983) was found also to promote the cell fusion in AC4. These results suggest that common regulatory mechanisms operate for sexual cell fusion among different mating systems in D. discoideum.     

The induction of nitrogenase activity in Rhizobium by non-legume plant cells

Summary Nitrogen fixation was induced in a strain of cowpea rhizobia, 32Hl, when it was grown in association with cell cultures of the non-legume, tobacco (Nicotiana tabacum). Rhizobia grown alone on the various media examined did not show nitrogenase activity, indicating the involvement of particular plant metabolites in nitrogenase induction. Nitrogenase activity, as measured by C₂H₂ reduction, was maximized at an O₂ concentration of 20% and at an assay temperature of 30°C, the conditions under which the plant cell-rhizobia associations developed. Glutamine, as a nitrogen source, could be replaced by other organic nitrogen sources, but NH₄ ⁺ and NO₃ ^- repressed nitrogenase activity. Nitrogenase activity induced in rhizobia when cultured adjacent to, but not in contact with, the plant cells could be stimulated by providing succinate in the medium. At least 12 other strains of rhizobia also reduced C₂H₂ in association with tobacco cells; the highest levels of activity were found among cowpea strains.