A Gene, ALCA, Affecting the Life Cycle Form Expressed in PHYSARUM POLYCEPHALUM

The usual sequence of forms in the Physarum polycephalum life cycle is plasmodium-spore-amoeba-plasmodium. So-called "amoebaless life cycle" or alc mutants of this Myxomycete undergo a simplified plasmodium-spore-plasmodium life cycle. We have analyzed three independently isolated alc mutants and found in each case that the failure of the spores to give rise to amoebae is due to a recessive Mendelian allele. The three mutations are tightly linked to one another and belong to a single complementation group, alcA. The mutations are pleiotropic, not only interfering with the establishment of the amoebal form at spore germination, but also affecting the phenotype of alc amoebae, which occasionally arise from alc spores. The alc amoebae (1) grow more slowly than wild type, particularly at elevated temperatures; (2) tend to transform directly into plasmodia, circumventing the sexual fusion of amoebae that usually accompanies plasmodium formation; and (3) form plasmodia by the sexual mechanism less efficiently than wild-type amoebae. The various effects of an alc mutation seem to derive from mutation of a single gene, since reversion for one effect is always accompanied by reversion for the other effects. Moreover, a mutation, aptA1, that blocks direct plasmodium formation by alcA amoebae, also increases their growth rate to near normal. The manner of plasmodium formation in alcA strains differs significantly from that in another class of mutants, the gad mutants. Unlike gad amoebae, alcA amoebae need not reach a critical density in order to differentiate directly into plasmodia and do not respond to the extracellular inducer of differentiation. In addition, alcA differentiation is not prevented by a mutation, npfA1, that blocks direct differentiation by most gad amoebae.     

Mutations Increasing Asexual Plasmodium Formation in PHYSARUM POLYCEPHALUM

Rare plasmodia formed in clones of heterothallic amoebae were analyzed in a search for mutations affecting plasmodium formation. The results show that the proportion of mutants varies with both temperature (18°, 26° or 30°) and mating-type allele (mt1, mt2, mt3, mt4). At one extreme, only one of 33 plasmoida formed by mt2 amoebae at 18° is mutant. At the other extreme, three of three plasmodia formed by mt1 amoebae at 30° are mutant. The mutant plasmodia fall into two groups, the GAD (greater asexual differentiation) mutants and the ALC (amoebaless life cycle) mutants. The spores of GAD mutants give rise to amoebae that differentiate into plasmodia asexually at much higher frequencies than normal heterothallic amoebae. Seven of eight gad mutations analyzed genetically are linked to mt and one (gad-12) is not. The gad-12 mutation is expressed in strains with different alleles of mt. The frequency of asexual plasmodium formation is heat sensitive in some (e.g., mt3 gad-11 ), heat-insensitive in two (mt2 gad-8 and mt2 gad-9) and cold-sensitive in one (mt1 gad-12) of twelve GAD mutants analyzed phenotypically. The spores of ALC mutants give rise to plasmodia directly, thereby circumventing the amoebal phase of the life cycle. Spores from five of the seven ALC mutants give rise to occasional amoebae, as well as plasmodia. The amoebae from one of the mutants carry a mutation (alc-1) that is unlinked to mt and is responsible for the ALC phenotype in this mutant. Like gad-12, alc-1 is expressed with different mt alleles. Preliminary observations with amoebae from the other four ALC mutants suggest that two are similar to the one containing alc-1; one gives rise to revertant amoebae, and one gives rise to amoebae carrying an alc mutation and a suppressor of the mutation.     

Analysis of development and growth in a mutant of Physarum polycephalum with defective cytokinesis

Cultures of amoebae of the mutant strain ATS23 isolated from strain CLd of Physarum polycephalum contain multinucleate cells and cells with increased nuclear DNA content. Plasmodia derived from ATS23 clones show abnormal morphology and defective sporulation. All abnormalities are enhanced by high incubation temperature (31 °C). Genetic analysis suggested that all the abnormalities were caused by a single mutation, denoted hts-23. The kinetics of plasmodium formation were followed in cultures of apogamic amoebae carrying hts-23 and hts⁺ (wild type) respectively. Results indicated that, relative to wild type, hts-23 did not increase the rate of plasmodium formation. There was evidence that, in both mutant and wild-type strains, commitment to plasmodium development occurred in uninucleate cells. Analysis of cell pedigrees by time-lapse cinematography indicated that the primary abnormal event in cultures of hts-23 amoebae was failure of cytokinesis; an apparently complete cleavage furrow was formed but cell separation failed, resulting in a binucleate cell. This event occurred randomly in pedigrees in which the majority of divisions were completed normally; its frequency increased during incubation at 31 °C. All other abnormalities in hts-23 amoebal cultures could be attributed to this primary event, assuming that DNA synthesis continued in the absence of cytokinesis and that the binucleate cells underwent the amoebal type of “open” mitosis, allowing the possibility of spindle fusion. This implies that the acquisition of “closed” mitosis is an essential early step in plasmodium development.     

Mutants with decreased differentiation to plasmodia in Physarum polycephalum

Summary Mutant (APT) amoebae that display reduced ability to form plasmodia asexually were isolated by the use of an enrichment procedure. The results of reconstruction experiments show that the procedure enriches only for mutants blocked early in the pathway from amoeba to plasmodium. Mutants were isolated from four parents, two of which produce plasmodia asexually because they carry the allele mth of the mating type locus, and two because they carry gad (greater asexual differentiation) mutations. The APT mutants varied widely in the frequency of residual plasmodium formation, which occurred, in some cases, by reversion. The mutants, called apt (amoeba to plasmodium transition), were recessive in diploids and linked to the mating type (mt) locus. Mutants derived from the gad parents, unlike the parents themselves, crossed readily with heterothallic amoebae. Progeny analysis from such crosses indicates that both gad mutations are linked to mt. The mutants derived from one of the mth parents fell into two groups on the basis of their ability to cross with the mutants derived from the mt2 gad-8 parent. The result suggests that the mth-derived mutants represent two or more complementation groups. Mutants derived from the mt2 gad-8 parent cross with mt2 amoebae and hence display an altered mating specificity.     

Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium     

Cellular events during sexual development from amoeba to plasmodium in the slime mould Physarum polycephalum

Time-lapse cinematography and immunofluorescence microscopy were used to study cellular events during amoebal fusions and sexual plasmodium development in Physarum polycephalum. Amoebal fusions occurred frequently in mixtures of strains heteroallelic or homoallelic for the mating-type locus matA, but plasmodia developed only in the matA-heteroallelic cultures. These observations confirmed that matA controls development of fusion cells rather than cell fusion. Analysis of cell pedigrees showed that, in both types of culture, amoebae fused at any stage of the cell cycle except mitosis. In matA-heteroallelic fusion cells, nuclear fusion occurred in interphase about 2 h after cell fusion; interphase nuclear fusion did not occur in matA-homoallelic fusion cells. The diploid zygote, formed by nuclear fusion in matA-heteroallelic fusion cells, entered an extended period of cell growth which ended in the formation of a binucleate plasmodium by mitosis without cytokinesis. In contrast, no extension to the cell cycle was observed in matA-homoallelic fusion cells and mitosis was always accompanied by cytokinesis. In matA-homoallelic cultures, many of the binucleate fusion cells split apart without mitosis, regenerating pairs of uninucleate amoebae; in the remaining fusion cells, the nuclei entered mitosis synchronously and spindle fusion sometimes occurred, giving rise to a variety of products. Immunofluorescence microscopy showed that matA-heteroallelic fusion cells possessed two amoebal microtubule organizing centres, and that most zygotes possessed only one; amoebal microtubule organization was lost gradually over several cell cycles. In matA-homoallelic cultures, all the cells retained amoebal microtubule organization.     

A New Mating Compatibility Locus in PHYSARUM POLYCEPHALUM

The rate and extent of plasmodium formation were studied in mating tests involving pairs of largely isogenic amoebal strains compatible for mating-type (mt) alleles. A systematic variability was observed: plasmodia formed either rapidly and extensively or slowly and inefficiently. Plasmodium formation was found to be 10³- to 10⁴-fold more extensive in "rapid" crosses than in "slow" crosses. A genetic analysis revealed that the variability reflects the influence of a multiallelic compatibility locus that determines mating efficiency. This compatibility locus (designated matB), together with the original mating type locus, mt (in this work designated matA), constitute a tetrapolar mating specificity system in Physarum polycephalum.     

Two Multiallelic Mating Compatibility Loci Separately Regulate Zygote Formation and Zygote Differentiation in the Myxomycete PHYSARUM POLYCEPHALUM

The mating of Physarum polycephalum amoebae, the ultimate consequence of which is a "plasmodium," was recently shown to be governed by two compatibility loci, matA (or mt) and matB (Dee 1978; Youngmanet al. 1979). We present evidence that matA and matB separately regulate two discrete stages of mating: in the first stage, amoebae (which are normally haploid) fuse in pairs, with a specificity determined by matB genotype, to form diploid zygotes; subsequent differentiation of the zygotes into plasmodia is regulated by matA and is unaffected by matB. Mixtures of amoebae carrying unlike matA and matB alleles formed diploids to the extent of 10 to 15% of the cells present, and the diploids differentiated into plasmodia. When only the matB alleles differed, diploid cells still formed to a comparable (5 to 10%) extent, but rather than differentiating, these diploids remained amoebae. When strains carried the same alleles of matB, formation of diploid cells was greatly reduced: in like-matB, like-matA mixtures, none of 320 cells examined was diploid; in like-matB, unlike mat-A mixtures, differentiating diploids could be detected, but at only 10(-3) to 10(-2) the frequency of unlike-matB, unlike-matA mixtures. The nondifferentiating diploid amoebae recovered from unlike-matB, like-matA mixtures were genetically stable through extensive growth, even though they grew more slowly than haploids (10-hr vs. 8-hr doubling period), and could be crossed with both haploids and diploids. The results of such higher ploidy and mixed ploidy crosses indicate that karyogamy does not invariably accompany zygote formation and differentiation.     

Growth and differentiation of wild type amoebae of Physarum polycephalum in liquid culture

A method for growing wild type amoebal strains of Physarum polycephalum in two-membered liquid culture is presented. The medium is a simple buffered salts solution. We found that a minimal level of divalent cation was required for growth. All amoebal strains tried to date have grown under our conditions in stationary culture. Growth under gyratory conditions was only successful at 60 rpm or less and consistent growth required a period of adaptation over several transfers. Differentiation of two apogamic strains, CL and CH1, were compared. Contrary to the results seen on agar plates, the time of onset for the first committed amoebae was identical for both strains in liquid culture. Attempts to demonstrate mating between two genetically compatible amoebal strains grown together in liquid culture were not successful.     

Mating type and the differentiated state in Physarum polycephalum.

In the acellular slime mold, Physarum polycephalum, the differentiation of amoebae into plasmodia is controlled by a mating type locus, mt. Amoebae carrying heterothallic alleles usually do not differentiate within clones; plasmodia form when two amoebae carrying different alleles fuse and undergo karyogamy. In this paper, we show that amoebae heterozygous for heterothallic alleles can be isolated and maintained as amoebae; the amoebae form plasmodia in clones without a change in ploidy. Plasmodia were also found to be formed, infrequently, by heterothallic amoebae of a single mating type. The plasmodia are healthy and are also formed without a change in ploidy. Thus, the presence of two different heterothallic mating type genes in a single nucleus is compatible with the amoebal state and one heterothallic mating type gene is compatible with the plasmodial state, once established.     

Karyotype polymorphism correlates with intraspecific infertility in the homothallic ascomycete Sordaria macrospora

The homothallic fungus Sordaria macrospora produces perithecia with meiotically derived ascospores. In most cases, intraspecies crosses between strains from different culture collections generate fertile hybrid perithecia in the contact zone of two mycelia. However, in some of these crosses we observed a significant decrease in the fertility of the hybrid perithecia when strains of different origin were used for mating. Since we assumed that chromosome variability between the culture collection strains might contribute to this reduction in fertility, we performed pulsed‐field gel electrophoresis. In the course of our study, we were able to identify two major groups of electrophoretic karyotypes in S. macrospora culture collection strains. A quantitative analysis revealed that polymorphic karyotypes contribute to a reduction of fertility in forced crosses between strains carrying differently sized chromosomes. The observed intraspecific chromosome length polymorphism might have consequences on the speciation process of a homothallic fungus capable of sexual but not of asexual spore formation.     

Revertants of selfing (gad) mutants in Physarum polycephalum

The conversion of the uninucleate amoebal form of Physarum polycephalum to the multi-nucleate plasmodial form is under the control of a genetic region which contains matA (or mt), a determinant of mating specificity. The region is the site of most gad mutations, which give amoebae the ability to produce plasmodia in clones without mating (ie, to self). In the present study, nonselfing revertants were isolated from two matA2-derived gad mutants and two matA3-derived gad mutants. Some revertants were found to have regained exactly, or nearly, the same phenotype as the original matA2 or matA3 strain. Others expressed new mating types, having gained the ability to mate with strains of the parental matA type. The results are compatible with a model in which new mating types arise from forward mutations (gad) and back mutations (npf or no plasmodium formation) occurring successively in a single gene, matA.     

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.     

Multiple Alleles and Other Factors Affecting Plasmodium Formation in the True Slime Mold Physarum polycephalum Schw*

SYNOPSIS. The life cycle of the true slime mold Physarum polycephalum includes 2 vegetative stages: the multinucleate coenocytic plasmodium and the uninucleate amoeba. A clone of amoebae established from a single spore does not normally yield plasmodia. Plasmodia are formed when amoebae from particular clones are mixed; thus plasmodium formation is said to be controlled by a ‘mating-type’ system. Previous work by the author with a sample of P. polycephalum derived from a single source revealed that 2 mating types were present and were determined by a pair of alleles at 1 locus. The present paper reveals the presence of 2 more mating types in a sample of P. polycephalum derived from a different source and provides evidence that these are determined by 2 alleles at the same locus as the other 2. Evidence for the presence of other inherited factors affecting plasmodium formation, the mode of action of these factors and possible explanations for the occurrence of plasmodia in single-spore cultures are also discussed.     

The specificity of Burkholderia symbionts in the social amoeba farming symbiosis: Prevalence,species, genetic and phenotypic diversity

The establishment of symbioses between eukaryotic hosts and bacterial symbionts in nature is a dynamic process. The formation of such relationships depends on the life history of both partners. Bacterial symbionts of amoebae may have unique evolutionary trajectories to the symbiont lifestyle, because bacteria are typically ingested as prey. To persist after ingestion, bacteria must first survive phagocytosis. In the social amoeba Dictyostelium discoideum, certain strains of Burkholderia bacteria are able to resist amoebal digestion and maintain a persistent relationship that includes carriage throughout the amoeba's social cycle that culminates in spore formation. Some Burkholderia strains allow their host to carry other bacteria, as food. This carried food is released in new environments in a trait called farming. To better understand the diversity and prevalence of Burkholderia symbionts and the traits they impart to their amoebae hosts, we first screened 700 natural isolates of D. discoideum and found 25% infected with Burkholderia. We next used a multilocus phylogenetic analysis and identified two independent transitions by Burkholderia to the symbiotic lifestyle. Finally, we tested the ability of 38 strains of Burkholderia from D. discoideum, as well as strains isolated from other sources, for traits relevant to symbiosis in D. discoideum. Only D. discoideum native isolates belonging to the Burkholderia agricolaris, B. hayleyella, and B. bonniea species were able to form persistent symbiotic associations with D. discoideum. The Burkholderia–Dictyostelium relationship provides a promising arena for further studies of the pathway to symbiosis in a unique system.     

Complementation of Amoebal-Plasmodial Transition Mutants in PHYSARUM POLYCEPHALUM

Amoebae of the Myxomycete Physarum polycephalum differentiate to yield plasmodia in two ways: in crossing, haploid amoebae of appropriate genotypes fuse to form diploid plasmodia; in selfing, plasmodia form without amoebal fusion or increase in ploidy. Amoebae carrying the mating-type allele matAh (formerly mt_h) self efficiently, but occasionally give rise to mutants that self at very low frequencies. Such "amoebal-plasmodial transition" mutants were mixed in pairs to test their ability to complement one another in the formation of plasmodia by crossing. The pattern of crossing permitted 33 mutants to be assigned to four complementation groups (aptA^-, npfA^-, npfB^- and npfC^-). Similar tests had previously proved only partially successful, as crossing had occurred only rarely in mixtures of compatible strains. The efficiency of complementation was greatly increased in the current work by mixing strains that carried different alleles of a newly-discovered mating-compatibility locus, matB; this locus had no effect on the specificity of complementation. A possible interpretation of the complementation behavior of the mutants is suggested.     

Mutagenic DNA repair in Escherichia coli. XVI. Mutagenesis by ultraviolet light plus delayed photoreversal in recA strains

Mutagenesis was demonstrable after delayed photoreversal of UV-irradiated strains carrying a recA deletion indicating that RecA protein is not essential for the misincorporation process that is revealed by delayed photoreversal. Moreover, the data suggest that RecA protein actually depresses misincorporation to varying extents depending on the recA allele. No delayed photoreversal was demonstrable in reA1 or recA56 bacteria unless the lexA102(ind-) allele was also present. It is suggested that the level of these RecA proteins may be lower in the lexA102(ind-) strains thus minimising their depressive effect. Delayed photoreversal mutagenesis in strains carrying the recA441 allele was not affected by either adenine or guanosine plus cytidine, substances which affect the proteolytic activity of RecA441 protein.     

Mutations affecting the initiation of plasmodial development in Physarum polycephalum

In the heterothallic myxomycete Physarum polycephalum, uninucleate amoebae normally differentiate into syncytial plasmodia following heterotypic mating. In order to study the genetic control of this developmental process, mutations affecting the amoebal-plasmodial transition have been sought. Numerous mutants characterized by self-fertility have been isolated. The use of alkylating mutagens increases the mutant frequency over the spontaneous level but does not alter the mutant spectrum. Three spontaneous and 14 induced mutants have been analyzed genetically. In each, the mutation appears to be linked to the mating type locus. In three randomly selected mutants, the nuclear DNA content is the same in amoebae and plasmodia, indicating that amoebal syngamy does not precede plasmodium development in these strains. These results indicate that a highly specific type of mutational event, occurring close to or within the mating type locus, can abolish the requirement for syngamy normally associated with plasmodial differentiation. These mutations help define a genomic region regulating the switch from amoebal to plasmodial growth.     

Marginal distribution and high heterozygosity of asexual Caloglossa vieillardii (Delesseriaceae,Rhodophyta) along the Australian coasts

In animals and land plants, many asexual species originate through inter‐ or intraspecific crosses, and such heterozygous asexuals frequently are more abundant than their sexual relatives in marginal habitats. Although asexual species have been reported in various macroalgal taxa, detailed information regarding their distribution, heterozygosity, and origin is limited. Because many asexual tetrasporophyte strains of Caloglossa vieillardii have been isolated from South Australia, far from their core tropical habitats, we re‐examined the distribution range of asexual C. vieillardii and genotyped these and other western Pacific strains using an actin gene marker. We confirmed the marginal distribution of the asexuals; however, a small patch of sexual thalli was newly discovered 450 km further west from asexual populations in South Australia. Three heterozygous genotypes and one homozygous genotypes were detected from nine asexual populations; 21 heterozygous strains were obligately asexual, but one homozygous strain suddenly produced sexual gametophytes after several years of culture. We hypothesized that the most abundant heterozygous genotype (defined as type 3/4) in asexual populations occurred by a cross between type 3 and type 4 allele gametophytes, both of which were isolated from the Australian coasts. In the crossing experiments, certain combinations between type 3 females and type 4 males produced tetrasporophytes, which recycled successive tetrasporophytes. In the culture experiments, whereas both sexual and asexual strains successfully produced tetraspores at 12°C, no sexual strains released carpospores below 14°C. However, it is uncertain whether this slight difference of maturation temperature was related to the marginal distribution of asexual C. vieillardii.     

The activation of cell aggregation by phosphorylation in Dictyostelium discoideum

Single amoebae of D. discoideum are phosphorylated in the presence of external ATP. Phosphorylation is catalyzed by a cAMP independent cell membrane bound protein kinase. As a result of phosphorylation cell aggregation is induced and the chemotactic sensitivity of the amoebae to a cAMP gradient decreased. Cell membrane phosphorylation may be involved in triggering cell aggregation in vivo. The fact that the number of free phosphorylable sites per cell decreases at the onset of aggregation gives support to this hypothesis. The existence of a plasma membrane bound phosphoprotein phosphatase suggests a possible regulator role for this enzyme on the phosphorylation of the amoebae. Finally, ATP inhibits intercellular contact sites outside the aggregation center. Despite this inhibiting effect on cell adhesiveness, amoebal movement toward an aggregation center maintains its normal periodicity.