Chemotactic response of wild-type and aggregation-defective mutants of Polysphondylium violaceum

Abstract. The aggregation-specific chemoattractant for Polysphondylium violaceum is N-propionyl-γ-L-glutamyl-L-ornithine-δ-lactam ethyl ester, or glorin. Wild-type amoebae allowed to develop in liquid culture acquire increased ability to respond to glorin shortly after starvation, i.e., just prior to the time they become aggregation competent. Similarly, as development proceeds, the amoebae show decreased sensitivity to folic acid, but they show almost no response to cyclic AMP at any time during their development in liquid culture. The optimum concentrations for the chemotactic response are 10^-8 M for glorin and 10^-5–10^-6 M for folic acid. A class of aggregation-defective mutants, aggA , will not aggregate in the absence of an excreted pheromone, D factor. During development in liquid culture in the presence or absence of D factor, these aggA mutants show a chemotactic response similar to that of wild-type amoebae to folic acid and glorin. However, D factor does enhance the chemotactic response of aggA mutants to glorin. In the absence of D factor, mutant amoebae will form fruiting bodies if exposed to a chemotactic gradient of either folic acid or glorin. Under these conditions, the mutant amoebae circumvent the requirement for D factor in order to develop.     

Founder Cell Differentiation and Acrasin Production in an Aggregateless Mutant of Polyspondylium violaceum

A specific class of aggregation-deficient mutants, aggA , of Polyshondylium violaceum are unable to aggregate unless supplied exogenously with a stimulating factor called D factor. The present study examines the effect of D factor on the induction of founder cells and on the production of the chemoattractant of aggregation, N-propionyl-γ-L-glutamyl-L-ornithine-δ-lactam ethyl ester (or glorin). Founder cells initiate aggregate formation and are morphologically distinct from the majority of the amoebae. Founder cell differentiation and oriented movement of attracted amoebae have been studied by time-lapse videotape analysis. In wild-type strains, on the average 90 min after the onset of starvation, a single, motile, irregularly shaped amoeba stops wandering and becomes round in shape. This founder cell has differentiated randomly from the pool of starved amoebae and within 2.5 min after the cessation of movement begins to attract and establish cellular contacts nighboring amoebae. The aggA mutants neither aggregate nor differentiate founder cells in the absence of D factor; whereas, aggregate formation and founder cell differentiation occur in the presence of physiological concentrations of purified, externally added D factor. However, in either the presence or absence of D factor, aggA amoebae produce and excrete glorin (measured using a bioassay) at levels comparable to their parental strain. These studies suggest that D factor is required for founder cell differentiation and organization of the aggregate, and that the ability to synthesize and excrete glorin is not sufficient to trigger aggregation.     

Extracellular cyclic AMP during development of the cellular slime mold Polysphondylium violaceum: comparison of accumulation in the wild type and an aggregation-defective mutant.

Cyclic AMP was synthesized by Polysphondylium violaceum after starvation and during the preaggregation stage of development. Most of the newly synthesized cyclic AMP accumulated in the extracellular medium, with very little change in the intracellular cyclic AMP concentration. The addition of 10(-3) to 10(-6) M exogenous cyclic AMP to starved amoebae caused a 20 to 50% decrease in the number of aggregation centers formed compared with untreated controls. An aggregation-defective mutant of P. violaceum (strain aggA586) excreted or accumulated very little cyclic AMP. Strain aggA586 aggregated normally in the presence of a dialyzable, excreted product (D factor) produced by wild-type amoebae. When the mutant was incubated with D factor, cyclic AMP accumulated in the medium, and the amount accumulated depended on the amount of D factor added to the mutant amoebae.     

Developmental regulation of production of an aggregation-stimulating factor from the cellular slime mold Polysphondylium violaceum

An excreted, dialyzable component(s) produced during development of wild-type Polysphondylium violaceum has been previously shown to stimulate aggregation of aggregateless mutants in the complementation group aggA. Production of this aggregation-stimulating factor, called D factor, is greater during development in liquid culture than during development on a surface. after partial purification of crude D factor using high performance liquid chromatography, multiple species are found that retain the ability to stimulate aggregation of the aggA mutants. The three major components (DfA, DfB, and DfC) show decreasing polarity based on purification using reverse-phase chromatography. The proportion of each component secreted varies, depending on the developmental conditions (surface versus liquid) and the time after starvation when the factors are isolated. Preliminary physical and chemical characterization of the three D factor components suggests that they are related.     

Purification, characterization, and partial structure of D factor from Polysphondylium violaceum

The A component of D factor (DfA) was overproduced during development of wild type Polyspondylium violaceum strain China after starvation in liquid medium. Crude DfA excreted by strain China was partially purified by ultrafiltration using Amicon YM10 and YM2 filters with DfA extracted from the filtrate by absorption onto a preparative grade C-18 resin. The concentrated material was further purified on a C-18 analytical column using both acetonitrile:water and methanol:water gradients. This highly purified fraction was a single component with a final specific activity of greater than 10(6) units per mg dry weight. Purified DfA is red having a broad visible absorbance at 500 nm and a ultraviolet (uv) absorbance at 290-300 nm. The red chromophore is sensitive to pH and to oxidation-reduction. 1H and 13C nmr studies with purified DfA indicate that it is a C11 compound with both polar and non-polar regions. The non-polar region has been identified as a hexanone and is the same as the side chain of DIF from Dictyostelium discoideum. Purified DfA has been used in studies with the D factor non-producing mutant, tsg-119 cyc-1 aggA586 (A586), to show that neither production of glorin nor chemotactic sensitivity to glorin are affected by D factor. However, founder cells develop in A586 mutant populations only after addition of D factor. These data suggest that DfA may be necessary for induction of aggregate formation by aggregation-competent amoebae.     

Analysis of developmentally defective chemical signaling mutants of Polysphondylium violaceum.

Six aggregation-defective mutants of Polysphondylium violaceum dependent on external addition of the pheromone D factor for aggregation were isolated after nitrosoguanidine mutagenesis. With a screening technique based on synergistic development, D-factor-dependent mutants can be separated from other kinds of aggregateless mutants. Genetic complementation analyses of the newly isolated mutants showed them to be mutant at the aggA locus. Individual mutants exhibited different sensitivities to D factor(s), responding maximally over a 300-fold range of concentrations.     

The cold war of the social amoebae

When confronted with starvation, the amoebae of Dictyostelium discoideum initiate a developmental process that begins with cell aggregation and ends with a ball of spores supported on a stalk. Spores live and stalk cells die. Because the multicellular organism is produced by cell aggregation and not by growth and division of a single cell, genetically diverse amoebae may enter an aggregate and, if one lineage has a capacity to avoid the stalk cell fate, it may have a selective advantage. Such cheater mutants have been found among wild isolates and created in laboratory strains. The mutants raise a number of questions--how did such a cooperative system evolve in the face of cheating? What is the basis of self recognition? What genes are involved? How is cheating constrained? This review summarizes the results of studies on the social behavior of Dictyostelium and its relatives, including the familiar asexual developmental cycle and the lesser known, but puzzling, sexual cycle.     

Genetic analysis of the aggA locus involved in agglutination and adherence of Pseudomonas putida, a beneficial fluorescent pseudomonad.

An isolate of Pseudomonas putida, which rapidly adheres to plant roots, is agglutinated by a glycoprotein from root surfaces. Agglutination is prevented and adherence to the root surface is diminished by Tn5 insertion in mutant 5123. Two cosmid clones from wild type P. putida and a 2.7-kbp EcoRI-HindIII subclone present in both cosmid clones restored agglutinable to wild type levels in transconjugants of the nonagglutinable (Agg-) Tn5 mutant 5123. These three clones increased agglutinability in transconjugants of the parental Agg+ isolate. The 2.7-kbp EcoRI-HindIII subclone restored adherence to bean root surfaces of 5123 to wild type levels in a short-term binding assay. Deletion analysis of the 2.7-kbp fragment indicated only 1.45 kbp was necessary for complementation of agglutinability in 5123. This sequence, termed the aggA locus, contains an open reading frame of 1,356 nucleotides encoding a predicted 50,509-Da protein. The distribution of the aggA locus in plant-associated bacteria, as detected through Southern hybridization, is limited to bacteria that express the agglutination phenotype.     

Competence-Independent Activity of Pneumococcal Enda Mediates Degradation of Extracellular DNA and Nets and Is Important for Virulence

Membrane surface localized endonuclease EndA of the pulmonary pathogen Streptococcus pneumoniae (pneumococcus) is required for both genetic transformation and virulence. Pneumococcus expresses EndA during growth. However, it has been reported that EndA has no access to external DNA when pneumococcal cells are not competent for genetic transformation, and thus, unable to degrade extracellular DNA. Here, by using both biochemical and genetic methods, we demonstrate the existence of EndA-mediated nucleolytic activity independent of the competence state of pneumococcal cells. Pneumococcal mutants that are genetically deficient in competence development and genetic transformation have extracellular nuclease activity comparable to their parental wild type, including their ability to degrade neutrophil extracellular traps (NETs). The autolysis deficient ΔlytA mutant and its isogenic choline-treated parental wild-type strain D39 degrade extracellular DNA readily, suggesting that partial cell autolysis is not required for DNA degradation. We show that EndA molecules are secreted into the culture medium during the growth of pneumococcal cells, and contribute substantially to competence-independent nucleolytic activity. The competence-independent activity of EndA is responsible for the rapid degradation of DNA and NETs, and is required for the full virulence of Streptococcus pneumoniae during lung infection.     

Formation of competent Bacillus subtilis cells.

The process of competent cell formation for transformation has been studied with early-stationary-phase (T1) cells of Bacillus subtilis which had been grown in an enriched Spizizen minimal medium and transferred to a second synthetic medium. Rifampin, chloramphenicol, and tunicamycin were strong inhibitors of competent cell formation, as well as vegetative growth. After formation, competent cells were no longer sensitive to the above agents. Methicillin and an inhibitor of chromosomal replication, hydroxyphenylazouracil, did not inhibit the development of competence. A D-alanine-requiring mutant strain developed competence even in the absence of D-alanine in the second medium. A T1-stage culture showed the activity of extracellular serine protease which is necessary for sporulation. Competent cell formation was completely blocked by 0.7 M ethanol, which is a specific inhibitor of early events during sporulation, including forespore septum formation. Competent cells were formed even in media which supported sporulation. The development of competence was also studied with spo0 mutants at 10 different loci. Most spo0 mutations repressed the development of competence except for spo0C, spo0G, and spo0J. These results suggest that competent cells are formed from early sporulating cells with the synthesis of cell wall materials and by factors whose genes are activated by the supply of nutrients. It is suggested that common steps are involved both in forespore septation and in competent cell formation.     

The proteinopathy of D169G and K263E mutants at the RNA Recognition Motif (RRM) domain of tar DNA-binding protein (tdp43) causing neurological disorders: A computational study

One of the multitasking proteins, transactive response DNA-binding protein 43 (tdp43) plays a key role in RNA regulation and the two pathogenic mutations such as D169G and K263E, located at the RNA Recognition Motif (RRM) of tdp43, are reported to cause neurological disorders such as Amyotrophic Lateral Sclerosis and FrontoTemporal Lobar Degeneration. As the exploration of the proteinopathy demands both structural and functional characterizations of mutants, a comparative analysis on the wild type and mutant tdp43 (D169G and K263E) and their complexes with RNA has been performed using computational approaches. Molecular dynamics simulations revealed comparatively stable mutant structures compared to wild type tdp43. Both mutants show lesser binding affinity toward RNA molecule when compared to the wild type tdp43. Some of the observed features, including the increased solvent-accessible surface area, conformational flexibility as well as unfolding of tdp43, and the altered RNA conformation in tp43-RNA complex, reveal the susceptibility of these mutants to induce conformational changes in tdp43 for a possible aggregation in the cytoplasm. Particularly, the enhanced aggregation propensity of both mutants also evidences the higher probability of cytoplasmic aggregation of tdp43 mutants. Hence, the present analysis highlighting the structural and functional aspects of wild and mutant tdp43 will form the basis to gain insight into the proteinopathy of tdp43 and the related structure-based drug discovery. Thus, tdp43 can be used as target to develop novel therapeutic approaches or drug designing.     

A temperature-sensitive adenylyl cyclase mutant of Dictyostelium

Dictyostelium development starts with the chemotactic aggregation of up to 10(6) amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a major segment of which had been amplified by error-prone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22 degrees C but not at 28 degrees C was characterized in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28 degrees C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP. Synergy experiments between wild-type and tsaca2 cells, shifted to the restrictive temperature at various stages of development, showed that ACA plays an important role in the control of cell sorting and tip formation.     

Developmental regulation of production of an aggregation-stimulating factor from the cellular slime mold Polysphondylirrm violaceurn

Abstract. An excreted, dialyzable component(s) produced during development of wild-type Polysphondylium violaceum has been previously shown to stimulate aggregation of aggregateless mutants in the complementation group agg A. Production of this aggregation-stimulating factor, called D factor, is greater during development in liquid culture than during development on a surface. After partial purification of crude D factor using high performance liquid chromatography, multiple species are found that retain the ability to stimulate aggregation of the agg A mutants. The three major components (Df_A, Df_B, and Df_C), show decreasing polarity based on purification using reverse-phase chromatography. The proportion of each component secreted varies, depending on the developmental conditions (surface versus liquid) and the time after starvation when the factors are isolated. Preliminary physical and chemical characterization of the three D factor components suggests that they are related.     

The regulation of cellular slime mold development: a factor causing development of Polysphondylium violaceum aggregation-defective mutants.

aggA mutants of Polysphondylium violaceum develop normally in synergistic mixtures with other aggregation-defective mutants. Cell to cell contact is not necessary for development. A small dialyzable factor(s) produced by wild-type and other aggregation-defective mutants triggers development of aggA mutants. This factor (D factor) is developmentally regulated, appearing early in development and then disappearing. Mutants require D factor until aggregation has just begun and then they can continue even in the absence of added factor. D factor is produced by many, but not all species of cellular slime molds and is developmentally regulated in Dictyostelium discoideum as well as P. violaceum.     

Tau protein liquid–liquid phase separation can initiate tau aggregation

The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid–liquid phase separation (LLPS) under cellular conditions and that phase‐separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho‐tau isolated from human Alzheimer brain. Droplet‐like tau can also be observed in neurons and other cells. We found that tau droplets become gel‐like in minutes, and over days start to spontaneously form thioflavin‐S‐positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.     

Biochemical characterization of a paraquat-tolerant mutant of Escherichia coli

The biochemical basis for paraquat tolerance was investigated using one of the paraquat-resistant Escherichia coli mutants previously isolated. When grown in the absence of paraquat (PQ2+), the specific activities of glucose-6-phosphate dehydrogenase and NADPH:PQ2+-diaphorase, both required for the expression of PQ2+ toxicity, were comparable in the wild type and the mutant. However, growth in the presence of 1 mM PQ2+ resulted in greater induction of these two enzymes in the wild type than in the mutant. Nevertheless, when the mutant was grown in 50 mM PQ2+, the activities of these two enzymes were comparable to those of the wild type grown in the presence of 1 mM PQ2+. Measurement of cyanide-resistant respiration, an indication of intracellular superoxide generation, showed that the intracellular flux of superoxide mediated by subsaturating concentrations of paraquat was significantly lower in the mutant than in the wild type. Extracellular superoxide formation, as measured by superoxide dismutase-inhibitable cytochrome c reduction, was higher in the wild type than in the mutant whether grown in the absence or the presence of PQ2+. The mutant did not show cross-resistance toward juglone or plumbagin, compounds known to exacerbate superoxide generation. The kinetics of [14C]PQ2+ uptake showed that the wild type accumulated PQ2+ against a concentration gradient, whereas the mutant seemed to do so only by facilitated diffusion. The results indicate that the impaired paraquat uptake system in the mutant results in the physiological and biochemical differences observed between the wild type and mutant.     

Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.     

Differential effects of Parkin and its mutants on protein aggregation, the ubiquitin-proteasome system, and neuronal cell death in human neuroblastoma cells

Mutations in Parkin, an E3 ligase, which participates in the ubiquitin-proteasome system (UPS), cause juvenile onset Parkinson's disease (PD). Some mutants aggregate upon over-expression, but the effects of such aggregation on the UPS and neuronal survival have not been characterized. We show in this study that transient over-expression of wild type (WT) Parkin or various mutants in human neuroblastoma cells leads to localized accumulation of green fluorescent protein (GFP(u)), an artificial proteasomal substrate, indicative of UPS dysfunction. Parkin mutants, but not WT, aggregated, and GFP(u) and ubiquitin accumulated within such aggregates. Apoptotic death occurred only with mutant Parkin over-expression, and correlated with aggregation, but not GFP(u) accumulation. Enzymatic proteasomal activity was slightly increased with WT Parkin and decreased with mutant Parkin over-expression. This decrease was, at least in part, due to caspase activation. We conclude that mutant forms of Parkin can exert toxic effects on neuronal cells, possibly through their propensity to aggregate. Both WT and mutant forms can induce localized UPS dysfunction, likely through different mechanisms. This raises a note of caution regarding forced over-expression of Parkin as a neuroprotective strategy in PD or other neurodegenerative conditions and suggests a possible toxic gain of function for certain mutant forms of Parkin.     

Distinctive features of the D101N and D101G variants of superoxide dismutase 1; two mutations that produce rapidly progressing motor neuron disease

Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis induce misfolding and aggregation of the protein with the inherent propensity of mutant SOD1 to aggregate generally correlating, with a few exceptions, to the duration of illness in patients with the same mutation. One notable exception was the D101N variant, which has been described as wild‐type‐like. The D101N mutation is associated with rapidly progressing motor neuron degeneration but shows a low propensity to aggregate. By assaying the kinetics of aggregation in a well‐characterized cultured cell model, we show that the D101N mutant is slower to initiate aggregation than the D101G mutant. In this cell system of protein over‐expression, both mutants were equally less able to acquire Zn than WT SOD1. In addition, both of these mutants were equivalently less able to fold into the trypsin‐resistant conformation that characterizes WT SOD1. A second major difference between the two mutants was that the D101N variant more efficiently formed a normal intramolecular disulfide bond. Overall, our findings demonstrate that the D101N and D101G variants exhibit clearly distinctive features, including a different rate of aggregation, and yet both are associated with rapidly progressing disease.

     

Replicative fitness of protease inhibitor-resistant mutants of human immunodeficiency virus type 1

The relative replicative fitness of human immunodeficiency virus type 1 (HIV-1) mutants selected by different protease inhibitors (PIs) in vivo was determined. Each mutant was compared to wild type (WT), NL4-3, in the absence of drugs by several methods, including clonal genotyping of cultures infected with two competing viral variants, kinetics of viral antigen production, and viral infectivity/virion particle ratios. A nelfinavir-selected protease D30N substitution substantially decreased replicative capacity relative to WT, while a saquinavir-selected L90M substitution moderately decreased fitness. The D30N mutant virus was also outcompeted by the L90M mutant in the absence of drugs. A major natural polymorphism of the HIV-1 protease, L63P, compensated well for the impairment of fitness caused by L90M but only slightly improved the fitness of D30N. Multiply substituted indinavir-selected mutants M46I/L63P/V82T/I84V and L10R/M46I/L63P/V82T/I84V were just as fit as WT. These results indicate that the mutations which are usually initially selected by nelfinavir and saquinavir, D30N and L90M, respectively, impair fitness. However, additional mutations may improve the replicative capacity of these and other drug-resistant mutants. Hypotheses based on the greater fitness impairment of the nelfinavir-selected D30N mutant are suggested to explain observations that prolonged responses to delayed salvage regimens, including alternate PIs, may be relatively common after nelfinavir failure.