Dormant Stages of the Green Flagellate Volvox in a Natural Habitat

Reproduction and formation of resistant dormant spores in Volvox aureus (Ehr.) and V. tertius (Meyer) were studied in a biocoenosis of a transient pool in July–August 1996 and 1997. Under these conditions, the populations of two Volvox species had species-specific reproductive features. In the V. tertius population, a relatively small amount of male individuals and dormant spores (zygospores) appeared sporadically. On the contrary, in the V. aureus population, dormant parthenospores were formed, but male individuals were never observed.     

Molecular phylogeny of the volvocine flagellates.

Phylogenetic studies of approximately 2,000 bases of sequence from the large and small nuclear-encoded ribosomal RNAs are used to investigate the origins of the genus Volvox. The colonial and multicellular genera currently placed in the family Volvocaceae form a monophyletic group that is significantly closer phylogenetically to Chlamydomonas reinhardtii than it is to the other unicellular green flagellates that were tested, including Chlamydomonas eugametos, Chlorella pyrenoidosa, and Haematococcus lacustris. Statistical analysis of 251 phylogenetically informative nucleotide positions rejects the "volvocine lineage" hypothesis, which postulates a monophyletic evolutionary progression from unicellular organisms (such as Chlamydomonas), through colonial organisms (e.g., Gonium, Pandorina, Eudorina, and Pleodorina) demonstrating increasing size, cell number, and tendency toward cellular differentiation, to multicellular organisms having fully differentiated somatic and reproductive cells (in the genus Volvox). The genus Volvox appears not to be monophyletic. Volvox capensis falls outside a lineage containing other representatives of Volvox (V. aureus, V. carteri, and V. obversus), and both of these Volvox lineages are more closely related to certain colonial genera than they are to each other. This implies either a diphyletic origin of Volvox from different colonial volvocacean ancestors, a phylogenetic derivation of some of the colonial genera from a multicellular (i.e., Volvox) ancestor, or both. Considered together with previously published observations, these results suggest that the different levels of organizational and developmental complexity found in the Volvocaceae represent alternative stable states, among which evolutionary transitions have occurred several times during the phylogenetic history of this group.     

Morphogenesis in the family Volvocaceae: different tactics for turning an embryo right-side out

Green algae of the family Volvocaceae provide an unrivalled opportunity to analyze an evolutionary pathway leading from unicellularity to multicellularity with division of labor. One key step required for achieving multicellularity in this group was the development of a process for turning an embryo inside out: a morphogenetic process that is now known as "inversion," and that is a diagnostic feature of the group. Inversion is essential because at the end of its embryonic cleavage divisions, each volvocacean embryo contains all of the cells that will be present in an adult, but the flagellar ends of all cells are pointed toward the interior, rather than toward the exterior where they will need to be to function in locomotion. Inversion has been studied in greatest detail in Volvox carteri, but although all other volvocacean species have to struggle with the same awkward situation of being wrong-side out at the end of cleavage, they do it in rather different ways. Here, the inversion processes of six different volvocacean species (Gonium pectorale, Pandorina morum, Eudorina unicocca, Volvox carteri, Volvox tertius, and Volvox globator) are compared, in order to illustrate the variation in inversion patterns that exists within this family. The simplest inversion process occurs in the plate-shaped alga Gonium pectorale and the most complicated in the spherical alga Volvox globator. Gonium pectorale goes only from a concave-bowl shape to a slightly convex plate. In Volvox globator, the posterior hemisphere inverts completely before the anterior pole opens and the anterior hemisphere slides over the already-inverted posterior hemisphere; during both halves of this inversion process, the regions of maximum cell-sheet curvature move progressively, as radially symmetrical waves, along the posterior-anterior axis.     

Effects of alpha- and beta-D-glucose on germination of spores of Bacillus megaterium QM B1551.

The effects of D-glucose anomers on the germination of dormant spores of Bacillus megaterium QM B1551 were studied, alpha-D-Glucose (1 mM) slightly initiated the germination of the dormant spores during 10 min incubation at 37 degrees C, while about 60% of the dormant spores became germinated with beta-D-glucose (1 mM) in the same conditions. From the above observations and the finding that only a trace amount of alpha- or beta-D-glucose may bind with the dormant spores, it is speculated that the beta-D-glucose-stereospecific receptor site for the germination exists on the surface of the dormant spores of the bacillus.     

Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores.

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Palindromic repetitive elements in the mitochondrial genome of Volvox

Group I introns were found in the cob and cox I genes of Volvox carteri. These introns contain tandem arrays of short palindromic sequences that are related to each other. Inspection of other regions in the mtDNA revealed that similar palindromic repetitive sequences are dispersed in the non-protein coding regions of the mitochondrial genome. Analysis of the group I intron in the cob gene of another member of Volvocaceae, Volvox aureus, has shown that its sequence is highly homologous to its counterpart in V. carteri with the exception of a cluster of palindromic sequences not found in V. carteri. This indicates that the palindromic clusters were inserted into the introns after divergence of the two species, presumably due to frequent insertions of the palindromic elements during evolution of the Volvocaceae. Possible involvement of the palindromic repetitive elements in the molecular evolution of functional RNAs is discussed.     

Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

Analysis of the slow germination of multiple individual superdormant Bacillus subtilis spores using multifocus Raman microspectroscopy and differential interference contrast microscopy

Aim: To analyse the dynamic germination of hundreds of individual superdormant (SD) Bacillus subtilis spores. Methods and Results: Germination of hundreds of individual SD B. subtilis spores with various germinants and under different conditions was followed by multifocus Raman microspectroscopy and differential interference contrast microscopy for 12 h and with temporal resolutions of ≤30 s. SD spores germinated poorly with the nutrient germinant used to isolate them and with alternate germinants targeting the germinant receptor (GR) used originally. The mean times following mixing of spores and nutrient germinants to initiate and complete fast release of Ca‐dipicolinic acid (CaDPA) (T_lag and T_release times, respectively) of SD spores were much longer than those of dormant spores. However, the ΔT_release times (T_release?T_lag) of SD spores were essentially identical to those of dormant spores. SD spores germinated almost as well as dormant spores with nutrient germinants targeting GRs different from the one used to isolate the SD spores and with CaDPA that does not trigger spore germination via GRs. Conclusions: Since (i) ΔT_release times were essentially identical in GR‐dependent germination of SD and dormant spores; (ii) rates of GR‐independent germination of SD and dormant spores were identical; (iii) large increases in T_lag times were the major difference in the GR‐dependent germination of SD as compared with spores; and (iv) higher GR levels are correlated with shorter T_lag times, these results are consistent with the hypothesis that low levels of a GR are the major reason that some spores in a population are SD with germinants targeting this same GR. Significance and Impact of the Study: This study provides information on the dynamic germination of individual SD spores and improves the understanding of spore superdormancy.     

Selective feeding and its effect on polymorphism and sexuality in the rotifer Asplanchna sieboldi

SUMMARY. Campanulate females of A. sieboldi (clone 12C1) attack Asplanchna brightwelli and A. girodi much more readily than either the alga Volvox aureus or the rotifer Brachionus calyciflorus . Pre-feeding campanulates on B. calyciflorus prior to testing does not appreciably affect their response to this prey. Discrimination during feeding occurs immediately after the campanulate's corona contacts a potential food organism and is probably mediated by chemoreceptors. Once a food organism is attacked, the probabilities of it being captured and subsequently swallowed are high and similar for each of the four organisms tested.
Since food organisms other than Asplanchna . such as V. aureus and B. calyciflorus , induce campanulates in this clone to produce cruciform, and thus frequently sexual, females in succeeding generations, a tendency of these campanulates to select congeneric prey would favour the maintenance of the campanulate morphotype and thus the continuation of parthenogenetic reproduction in the ensuing population.     

Content, distribution and stability of protein-synthesis elongation factor Tu in subcellular fractions of vegetative cells and spores of Streptomyces aureofaciens

The protein synthesis elongation factor Tu (EF-Tu) was identified in dormant spores of Streptomyces aureofaciens and its content and distribution in vegetative cells and dormant spores were determined. Cell-free homogenates from spores were found to contain a EF-Tu cleaving membrane bound protease. The protease cleaved aggregated EF-Tu much less efficiently than non-aggregated factor in cell homogenates. The relative content of EF-Tu and ribosomes in dormant spores was very similar to that found in exponentially growing vegetative cells.     

Decreased particulate NADH oxidase activity in Bacillus subtilis spores after polymyxin B treatment

The activities of several enzymes of polymyxin B-treated dormant and germinated spores of Bacillus subtilis were examined. The particulate NADH oxidase of the antibiotic-treated spores showed considerably lower specific and total activities compared with those of untreated ones. The specific and total NADH oxidase activities of untreated spores increased 12- and 15-fold respectively during germination, whereas increases during germination of polymyxin B-treated spores were inhibited. The specific and total activities of particulate NADH cytochrome c reductase of dormant spores were decreased by polymyxin B treatment in almost the same proportion as those of the particulate NADH oxidase. The specific activity of NADH dehydrogenase of dormant spores remained unchanged after antibiotic treatment but the total activity fell considerably. The activities of other enzymes examined were similar for untreated dormant and germinated spores and antibiotic-treated spores. The respiration of polymyxin B-treated dormant spores was inhibited at the same time as the start of germination. Morphologically, polymyxin B-treated dormant spores lost a laminar structure of the cortex and details of the spore protoplast. The inhibitory mechanism of particulate NADH oxidase activity of polymyxin B-treated dormant spores is discussed.     

Fixation of Dormant Tilletia Teliospores for thin Sectioning

Dormant Tilletia caries teliospores in fixative solution or distilled water were frozen onto specimen chucks of an FTS Sorvall- Christensen frozen thin sectioner and cut or fractured at various temperatures (-20 C to -75 C) and thickness settings (10, 15, 20, and 25 μm). Cytoplasm of dormant spores was well preserved and organelles were found to differ from those of germinated spores in morphology. This procedure makes it possible to fix adequately dormant spores and thus compare the ultrastructurc and histochemistry of dormant spores with those of germinated spores.     

Analysis of dye binding by and membrane potential in spores of Bacillus species

Aims:  To determine roles of coats in staining Bacillus subtilis spores, and whether spores have membrane potential.
Methods and Results:  Staining by four dyes and autofluorescence of B. subtilis spores that lack some ( cotE , gerE ) or most ( cotE gerE) coat protein was measured. Wild-type, cotE and gerE spores autofluorescenced and bound dyes, but cotE gerE spores did not autofluorescence and were stained only by two dyes. A membrane potential-sensitive dye DiOC₆(3) bound to dormant Bacillus megaterium and B. subtilis spores. While this binding was abolished by the protonophore FCCP, DiOC₆(3) bound to heat-killed spores, but not to dormant B. subtilis cotE gerE spores. However, DiOC₆(3) bound well to all germinated spores.
Conclusions:  The autofluorescence of dormant B. subtilis spores and the binding of some dyes are due to the coat. There is no membrane potential in dormant Bacillus spores, although membrane potential is generated when spores germinate.
Significance and Impact of the Study:  The elimination of the autofluorescence of B. subtilis spores may allow assessment of the location of low abundance spore proteins using fluorescent reporter technology. The dormant spore's lack of membrane potential may allow tests of spore viability by assessing membrane potential in germinating spores.     

The Protoplasmic Connection in Volvox

SYNOPSIS. Electron-microscopic observations were performed on 2 species of Volvox , one similar to V. globator , the other to V. aureus. The former has distinct protoplasmic connections in the adult coenobium and specific structures, named "medial bodies," in the connections just at the intersection with the middle lamella. The medial body is disk shaped, about 800 mμ in diameter, and is composed of 3 parts, 2 dense outer layers and an intermediate less dense zone. In the latter species, the connection and medial body were not seen. On the other hand, it was commonly seen in both of them that in younger, dividing gonidia neighboring protoplasts were connected with each other by protoplasmic bridges. The bridges are undoubtedly formed due to incomplete cell separation in the division of a gonidium. The structural difference in the adult coen***bium between the 2 species emerges just after inversion of the coenobium. In the globator type the medial body appears just after inversion, and the connection remains unruptured all thru life. In the aureus type, it seems that the connections are withdrawn or degenerate immediately after inversion. It is discussed whether protoplasmic continuity is really maintained by the connection or not in the freeswimming coenobium of Volvox.     

A Method for Extracting RNA from Dormant and Germinating Bacillus subtilis Strain 168 Endospores

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid–phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10⁸ spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.     

Fixation of Dormant Tilletia Teliospores for thin Sectioning

Dormant Tilletia caries teliospores in fixative solution or distilled water were frozen onto specimen chucks of an FTS Sorvall- Christensen frozen thin sectioner and cut or fractured at various temperatures (-20 C to -75 C) and thickness settings (10, 15, 20, and 25 μm). Cytoplasm of dormant spores was well preserved and organelles were found to differ from those of germinated spores in morphology. This procedure makes it possible to fix adequately dormant spores and thus compare the ultrastructurc and histochemistry of dormant spores with those of germinated spores.     

TEMPERATURE DEPENDENCE OF GROWTH AND PHOSPHORUS UPTAKE IN TWO SPECIES OF VOLVOX (VOLVOCALES,CHLOROPHYTA)1

The growth of Volvox globator L. and Volvox aureus Ehr. was measured at five temperatures and nine phosphorus concentrations. Growth rates were hyperbolically related to phosphorus concentrations for all temperatures using a Monod growth model. Optimal growth rates of 1.17 and 1.00 doublings d^?1 were obtained at 20°C for V. globator and V. aureus, respectively. Neither species grew at 5°C. The half-saturation constants for growth, K_s, were lower for V. aureus. Phosphorus uptake by both species was also dependent upon external phosphorus concentrations and temperature. At all temperatures, maximum phosphorus uptake (μmol P colony^?1 min^?1) was similar for both species; however, the half-saturation constants for uptake showed significant differences between the species. Comparisons of the kinetic constants for growth and phosphorus uptake suggest that V. aureus will outcompete V. globator under phosphorus limited, conditions.