Studies of cell-surface glorin receptors, glorin degradation, and glorin-induced cellular responses during development of Polysphondylium violaceum

The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethylester (glorin). Here we examine the binding properties of tritiated glorin to intact P. violaceum cells. Scatchard analysis of binding data yielded slightly curvilinear plots with Kd values in the range of 20 and 100 nM. The number of glorin receptors increased from 35,000 in the vegetative stage to 45,000 per cell during aggregation. Later, during culmination receptor numbers decreased to undetectable levels (less than 1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 degrees C, and ligand dissociation occurs from two kinetically distinct receptors whose half-times were 2 s for 72% of the bound glorin and 28 s for the remainder. The enzymatic degradation of glorin did not affect binding data during incubations of up to 1 min at 0 degrees C. Two glorinase activities were observed. An ornithine delta-lactam cleaving activity with a Km of ca. 10(-4) M and a propionic acid removing activity (Km 10(-5) M), both of which were detected mainly on the cell surface. Cleavage of the lactam occurred at a higher rate than removal of propionic acid. Lactam-cleaved glorin showed no chemotactic activity nor did it bind to cell-surface glorin receptors. Cell-surface-bound glorinase activity and glorin-induced cGMP synthesis were developmentally regulated, peaking at aggregation. In the most sensitive stage half-maximal responses (cGMP synthesis, chemotaxis, light-scattering) were elicited in the 10-100 nM range. Neither cAMP synthesis nor glorin-induced glorin synthesis was observed. Guanine nucleotides specifically modulated glorin receptor binding on isolated membranes, and, conversely, glorin modulated GTP gamma S binding to membrane preparations. Our results support the notion that glorin mediates chemotactic cell aggregation in P. violaceum acting via cell-surface receptors, G-proteins, and cGMP accumulation.     

Glorin-regulated protein synthesis in Polyspondylium violaceum

Protein synthesis by Polyspondylium violaceum in response to the chemoattractant glorin was analyzed by two-dimensional gel electrophoresis to determine if glorin can affect gene expression. When cells developing in a shaking suspension culture were given glorin, five proteins exhibited significantly increased and three proteins exhibited decreased incorporation of -[³⁵S]methionine. Glorin was active from 10–1000 nM, a concentration range within which cells are chemotactically active. The extent of the response was dependent on the concentration and the length of exposure to glorin. This evidence suggests that glorin may act in part to mediate changes in gene expression during development.     

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.     

Glorin-regulated protein synthesis in Polyspondylium violaceum

Protein synthesis by Polyspondylium violaceum in response to the chemoattractant glorin was analyzed by two-dimensional gel electrophoresis to determine if glorin can affect gene expression. When cells developing in a shaking suspension culture were given glorin, five proteins exhibited significantly increased and three proteins exhibited decreased incorporation of L-[35S]methionine. Glorin was active from 10-1000 nM, a concentration range within which cells are chemotactically active. The extent of the response was dependent on the concentration and the length of exposure to glorin. This evidence suggests that glorin may act in part to mediate changes in gene expression during development.     

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.     

Developmental gene regulation by an ancient intercellular communication system in social amoebae

The social amoebae (Dictyostelia) use quorum sensing-like communication systems to coordinate the periodic transition from uni- to multicellularity. The monophyletic descent of the Dictyostelia provides a unique opportunity to study the origin and adaptive evolution of such intercellular communication systems. We determined that the ability of aggregation-competent cells to respond to the intercellular messenger glorin occurred in the most ancient taxa of the Dictyostelia. We show using Illumina sequencing technology that glorin mediates rapid changes in gene expression at the transition from vegetative growth to aggregation. We conclude that peptide-based communication is the most ancient form of intercellular signaling in the evolution of multicellularity in the social amoebae, but has been repeatedly replaced by other communication systems during the monophyletic evolution of the social amoebae. Glorin communication has parallels with quorum sensing in that the molecule diffuses into the field, stimulates gene expression in receptive cells and coordinates a population-wide response.     

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.     

Whorl Formation in Polysphondylium violaceum: Relevance to Organization by Cyclic AMP

Whorl formation in Polysphondylium is a simple and good system for the study of pattern formation. The first step of whorl formation is characterized by separation of the rear cell mass from an advancing primary mass during culmination. Using the iontophoresis method, it has been shown that after the establishment of multicellular organization cells respond chemotactically to 3',5' -cyclic adenosine monophosphate (cAMP), but not to glorin, the chemoattaractant at the aggregating stage. P. violaceum cell masses were also found to secrete actively cAMP. Therefore, morphogenetic movement of P. violaceum cells after aggregation could be controlled mainly by the cAMP signalling system. Vital staining of cells with neutral red (NR) revealed that there are anterior-like cells stained well with NR in the posterior region of a migrating P. violaceum slug, and that the staining pattern changes markedly during whorl formation. Just before separation of the whorl mass, the anterior-like cells altered their distribution, and eventually were arranged as an equatorial band on the surface of the presumptive whorl mass, which probably would turn to organizing tip cells of the secondary masses left on the primary stalk. Thus whorl formation may be caused by separation of the strongest cAMP-source into two regions; the primary (anterior) and secondary (posterior) tips.     

Revision of the Australian species of the weevil genus Trigonopterus Fauvel

The Australian species of the genus Trigonopterus Fauvel are revised. Eight previously recognized species are redescribed and 24 additional new species are described: Trigonopterus allaetus Riedel, sp. n., Trigonopterus athertonensis Riedel, sp. n., Trigonopterus australinasutus Riedel, sp. n., Trigonopterus australis Riedel, sp. n., Trigonopterus bisignatus Riedel, sp. n., Trigonopterus bisinuatus Riedel, sp. n., Trigonopterus boolbunensis Riedel, sp. n., Trigonopterus cooktownensis Riedel, sp. n., Trigonopterus daintreensis Riedel, sp. n., Trigonopterus deplanatus Riedel, sp. n., Trigonopterus finniganensis Riedel, sp. n., Trigonopterus fraterculus Riedel, sp. n., Trigonopterus garradungensis Riedel, sp. n., Trigonopterus hasenpuschi Riedel, sp. n., Trigonopterus hartleyensis Riedel, sp. n., Trigonopterus kurandensis Riedel, sp. n., Trigonopterus lewisensis Riedel, sp. n., Trigonopterus montanus Riedel, sp. n., Trigonopterus monteithi Riedel, sp. n., Trigonopterus mossmanensis Riedel, sp. n., Trigonopterus oberprieleri Riedel, sp. n., Trigonopterus robertsi Riedel, sp. n., Trigonopterus terraereginae Riedel, sp. n., Trigonopterus yorkensis Riedel, sp. n.. All new species are authored by the taxonomist-in-charge, Alexander Riedel. Lectotypes are designated for the following names: Idotasia aequalis Pascoe, Idotasia albidosparsa Lea, Idotasia evanida Pascoe, Idotasia laeta Lea, Idotasia rostralis Lea, Idotasia sculptirostris Lea, Idotasia squamosa Lea. A new combination of the name Idotasia striatipennis Lea is proposed: Trigonopterus striatipennis (Lea), comb. n.. A key to the species is provided. Australian Trigonopterus occur in coastal Queensland, narrowly crossing into New South Wales. The southern parts of the range are inhabited by species found on foliage. A rich fauna of 19 edaphic species inhabiting the leaf litter of tropical forests is reported for the first time from the Australian Wet Tropics.     

Organization of the nar genes at the chlZ locus

The two membrane-bound respiratory nitrate reductases of Escherichia coli are encoded by distinct operons at two different loci, chlC and chlZ, on the chromosome. The chlZ locus includes a narK homologue, narU, encoding a nitrite extrusion protein, and narZYWV encoding nitrate reductase Z. No apparent homologue to the narXL operon has been found. Homology between narU and narK on the one hand and narZYWV and narGHJI on the other hand is limited to the coding regions.     

Influence of the acoustic environment on the distribution of the frog Ranidella riparia

At its southern edge the distribution of the frog Ranidella riparia abuts, with only a narrow zone of overlap, that of its allopatric sibling species R. signifera. In previous reports there was no evidence for any reduced survival of R. riparia in the creeks occupied by R. signifera immediately adjacent to the edge of its distribution. Here we examine the hypothesis that the acoustic environment in those creeks might inhibit successful communication by R. riparia. Although the structural characteristics of the R. riparia call were less suitable than those of R. signifera for transmission through the heavily vegetated habitat characteristic of those creeks, this alone did not inhibit successful reproduction by R. riparia. At a distance of 25 cm the average intensity of a call from an R. riparia male was 24 dB lower than one from R. signifera. In addition R. signifera form a continuous chorus in which the minimum sound intensity always exceeds that of single R. riparia calls at 25 cm. We propose that R. riparia cannot colonize creeks adjacent to their present distribution because the loud noise from R. signifera choruses either suppresses their calling activity, or makes them inaudible to potentially receptive females.     

Evidence of biosynthetic simplicity in the flavonoid chemistry of the ricciaceae

The major flavonoids in Riccia crystallina are naringenin and its 7-O-glucoside, apigenin 7-O-glucoside and apigenin 7-O-glucuronide and derivatives. Ricciocarpus natans is a rich source of luteolin 7,3′-di-O-glucuronide and also contains the 7-O-glucuronides of apigenin and luteolin and the 3′-O-glucuronide of luteolin. A parallel between the production of biosynthetically simple flavonoids and reduced morphology is evident among these liverworts.     

On the occurrence of tuliposides in the liliiflorae

Approximately 200 samples of liliiflorous plants were investigated for the presence of tuliposides. Appreciable amounts of tuliposide A were detected in all species of Erythronium, Tulipa, Gagea, Bomarea and Alstroemeria. Large amounts of tuliposide B seem to be restricted to Erythronium and Tulipa. The occurrence of identical post-inhibitins in Tulipa and allied taxa and in Alstroemeria and allied taxa is interpreted as indicating a close relationship between Lilioideae and Alstroemeriaceae. At the same time the allergenic potentialities of all taxa of Alstroemeria are stressed.     

Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase

F_OF₁ ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of F_OF₁. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. b_I and b_II). The results show the b dimer to be located at a non-catalytic α/β cleft, with b_I close to subunit α, whereas b_II is proximal to subunit β. Furthermore, b_I can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-b_I-α, b_II-β, α-b_I-b_II-β, and a-b_I-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that b_I is functionally related to the single b subunit present in mitochondrial ATP synthase.     

Distribution of Bacillus thuringiensis in the complex of spore-forming bacteria of the genus Bacillus Cohn in the soils of the nut-fruit forests of southern Kyrgyzstan

Three-year-long investigation of the microflora in the soil of nut-fruit forests in the south of Kyrgyzstan showed a wide distribution of spore-forming bacteria Bacillus subtilis, B. cereus, B. idosus, and B. megaterium. In the complex of these bacteria, crystal-forming bacteria B. thuringiensis are abundant. The occurrence of B. thuringiensis in mountain-forest black-brown soils reaches 80% of soil samples. Dominating subspecies are tohokuensis (H17), israelensis (H14), and toguchini (H31). Abundance of B. thuringiensis was insignificant and accounted for only 1% of spore-forming bacteria. The abundance B. thuringiensis in oozy biocenoses on the shores of water bodies within the Sary-Chelek Biosphere Reserve reaches 12.6–18.0% of soil-forming bacteria. Thus, it is possible to assume that B. thuringiensis not only are conserved in soils but also can reproduce.     

Checklist of the species of the aseptate Gregarine family Urosporidae

A checklist is given of the 45 named species of the family Urosporidae (phylum Protozoa, subphylum Apicomplexa, class Sporozoa, subclass Gregarinia, order Eugregarinida, suborder Aseptatina) together with their synonyms, the names of their hosts, their locations in the hosts, their known geographic distribution, and key references. Another list is given of synonyms, lapsi calami, nomina nuda, etc. associated with the genera of this family. The following taxonomic-nomenclatural innovations are introduced—NEW NAME: Gonospora good-richae nom. nov. in the polychaete Arenicola ecaudata; NEW COMBINATIONS: Urospora grassei (Changeux, 1961) in the sea cucumbers Holothuria spp.; Urospora schneideri (Mingazzini, 1891) in the sea cucumbers Holothuria spp; Gonospora gonadipertha (Djakonov, 1923) in the sea cucumber Cucumaria frondosa; Gonospora stichopi (Lützen, 1967) in the sea cucumber Stichopus tremulus.     

Role of hypermutability in the evolution of the genus Oenococcus

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium primarily responsible for malolactic fermentation in wine. A recent comparative genomic analysis of O. oeni PSU-1 with other sequenced lactic acid bacteria indicates that PSU-1 lacks the mismatch repair (MMR) genes mutS and mutL. Consistent with the lack of MMR, mutation rates for O. oeni PSU-1 and a second oenococcal species, O. kitaharae, were higher than those observed for neighboring taxa, Pediococcus pentosaceus and Leuconostoc mesenteroides. Sequence analysis of the rpoB mutations in rifampin-resistant strains from both oenococcal species revealed a high percentage of transition mutations, a result indicative of the lack of MMR. An analysis of common alleles in the two sequenced O. oeni strains, PSU-1 and BAA-1163, also revealed a significantly higher level of transition substitutions than were observed in other Lactobacillales species. These results suggest that the genus Oenococcus is hypermutable due to the loss of mutS and mutL, which occurred with the divergence away from the neighboring Leuconostoc branch. The hypermutable status of the genus Oenococcus explains the observed high level of allelic polymorphism among known O. oeni isolates and likely contributed to the unique adaptation of this genus to acidic and alcoholic environments.     

Evidence of the Insensitivity of the alpha-inc Allele to the Function of the Homothallic Genes in Saccharomyces Yeasts

The possible function of the α-inc allele (an α mating-type allele that is insensitive to the function of the homothallic gene system) was investigated by means of protoplast fusion. The fusion of protoplasts prepared from haploid strains of α-inc HO HMα HMa and α ho hmα HMa gave rise mainly to nonmating clones (58 of 64 isolates) and a few clones (six of 64 isolates) showing α mating type. Thirty of the 58 nonmating clones showed the diploid cell size and 28 clones had a larger cell size. Tetrad analysis of the nonmating clones with diploid cell size indicated that they were a/α-inc diploid; the normal α allele in α/α-inc cells was preferentially switched to an a allele. This observation further indicated that the HO/ho HMα/hmα HMa/HMa genotype is effective for the conversion of the α to a and that the inconvertibility of the α-inc allele is due to the insensitivity of the mating-type allele to the functional combination of the homothallic genes. It was suspected that fusion products larger than diploid cells might have been caused by multiple fusion of protoplasts.     

Phylogenetic analysis of the genus Pseudoroegneria and the Triticeae tribe using the rbcL gene

The Pseudoroegneria species are perennial grasses in the Triticeae tribe, whose St genome has been linked to several important polyploid species. Due to frequent hybridization and complex genetic mechanism, the relationships within Pseudoroegneria, and within the Triticeae have been heavily disputed. Using the chloroplast rbcL gene we estimated the nucleotide diversity of 8 Pseudoroegneria species. We also examined the phylogenetic relationships within Pseudoroegneria and of Pseudoroegneria within the Triticeae. The estimates of nucleotide diversity indicated that Pseudoroegneria tauri and Pseudoroegneria spicata species had the highest diversity, while Pseudoroegneria gracillima had the lowest diversity. The phylogenetic analysis of Pseudoroegneria placed all P. spicata species into a clade separate from the other Pseudoroegneria species, while the relationship of the other Pseudoroegneria species could not be determined. Due to the groupings of Pseudoroegneria with the polyploid Elymus, our results strongly supported Pseudoroegneria as the maternal genome donor to Elymus. There was also weak support that P. spicata may be the maternal donor to the StH Elymus species.