Polytoma Research Articles

The plastomes of Hyalomonas oviformis and Hyalogonium fusiforme evolved dissimilar architectures after the loss of photosynthesis.

The loss of photosynthesis in land plants and algae is typically associated with parasitism but can also occur in free‐living species, including chlamydomonadalean green algae. The plastid genomes (ptDNAs) of colorless chlamydomonadaleans are surprisingly diverse in architecture, including highly expanded forms ( Polytoma uvella and Leontynka pallida) as well as outright genome loss (Polytomella species). Here, we explore the ptDNAs of Hyalomonas (Hm.) oviformis (SAG 62‐27; formerly known as Polytoma oviforme) and Hyalogonium (Hg.) fusiforme (SAG 62‐1c), each representing independent losses of photosynthesis within the Chlamydophyceae. The Hm. oviformis ptDNA is moderately sized (132 kb) with a reduced gene complement (but still encoding the ATPase subunits) and is in fact smaller than that of its photosynthetic relative Hyalomonas chlamydogama SAG 11‐48b (198.3 kb). The Hg. fusiforme plastome, however, is the largest yet observed in nonphotosynthetic plants or algae (~463 kb) and has a coding repertoire that is almost identical to that of its photosynthetic relatives in the genus Chlorogonium. Furthermore, the ptDNA of Hg. fusiforme shows no clear evidence of pseudogenization, which is consistent with our analyses showing that Hg. fusiforme is the nonphotosynthetic lineage of most recent origin among known colorless Chlamydophyceae. Together, these new ptDNAs clearly show that, in contrast to parasitic algae, plastid genome compaction is not an obligatory route following the loss of photosynthesis in free‐living algae, and that certain chlamydomonadalean algae have a remarkable propensity for genomic expansion, which can persist regardless of the trophic strategy.

The plastid genomes of nonphotosynthetic algae are not so small after all

ABSTRACTThe thing about plastid genomes in nonphotosynthetic plants and algae is that they are usually very small and highly compact. This is not surprising: a heterotrophic existence means that genes for photosynthesis can be easily discarded. But the loss of photosynthesis cannot explain why the plastomes of heterotrophs are so often depauperate in noncoding DNA. If plastid genomes from photosynthetic taxa can span the gamut of compactness, why can't those of nonphotosynthetic species? Well, recently we showed that they can. The free-living, heterotrophic green alga Polytoma uvella has a plastid genome boasting more than 165 kilobases of noncoding DNA, making it the most bloated plastome yet found in a heterotroph. In this addendum to the primary study, we elaborate on why the P. uvella plastome is so inflated, discussing the potential impact of a free-living vs. parasitic lifestyle on plastid genome expansion in nonphotosynthetic lineages.

The Plastid Genome of Polytoma uvella Is the Largest Known among Colorless Algae and Plants and Reflects Contrasting Evolutionary Paths to Nonphotosynthetic Lifestyles.

The loss of photosynthesis is frequently associated with parasitic or pathogenic lifestyles, but it also can occur in free-living, plastid-bearing lineages. A common consequence of becoming nonphotosynthetic is the reduction in size and gene content of the plastid genome. In exceptional circumstances, it can even result in the complete loss of the plastid DNA (ptDNA) and its associated gene expression system, as reported recently in several lineages, including the nonphotosynthetic green algal genus Polytomella Closely related to Polytomella is the polyphyletic genus Polytoma, the members of which lost photosynthesis independently of Polytomella Species from both genera are free-living organisms that contain nonphotosynthetic plastids, but unlike Polytomella, Polytoma members have retained a genome in their colorless plastid. Here, we present the plastid genome of Polytoma uvella: to our knowledge, the first report of ptDNA from a nonphotosynthetic chlamydomonadalean alga. The P. uvella ptDNA contains 25 protein-coding genes, most of which are related to gene expression and none are connected to photosynthesis. However, despite its reduced coding capacity, the P. uvella ptDNA is inflated with short repeats and is tens of kilobases larger than the ptDNAs of its closest known photosynthetic relatives, Chlamydomonas leiostraca and Chlamydomonas applanata In fact, at approximately 230 kb, the ptDNA of P. uvella represents the largest plastid genome currently reported from a nonphotosynthetic alga or plant. Overall, the P. uvella and Polytomella plastid genomes reveal two very different evolutionary paths following the loss of photosynthesis: expansion and complete deletion, respectively. We hypothesize that recombination-based DNA-repair mechanisms are at least partially responsible for the different evolutionary outcomes observed in such closely related nonphotosynthetic algae.

Accelerated evolution of functional plastid rRNA and elongation factor genes due to reduced protein synthetic load after the loss of photosynthesis in the chlorophyte alga Polytoma.

Polytoma obtusum and Polytoma uvella are members of a clade of nonphotosynthetic chlorophyte algae closely related to Chlamydomonas humicola and other photosynthetic members of the Chlamydomonadaceae. Descended from a nonphotosynthetic mutant, these obligate heterotrophs retain a plastid (leucoplast) with a functional protein synthetic system, and a plastid genome (lpDNA) with functional genes encoding proteins required for transcription and translation. Comparative studies of the evolution of genes in chloroplasts and leucoplasts can identify modes of selection acting on the plastid genome. Two plastid genes--rrn16, encoding the plastid small-subunit rRNA, and tufA, encoding elongation factor Tu--retain their functions in protein synthesis after the loss of photosynthesis in two nonphotosynthetic Polytoma clades but show a substantially accelerated rate of base substitution in the P. uvella clade. The accelerated evolution of tufA is due, at least partly, to relaxed codon bias favoring codons that can be read without wobble, mainly in three amino acids. Selection for these codons may be relaxed because leucoplasts are required to synthesize fewer protein molecules per unit time than are chloroplasts (reduced protein synthetic load) and thus require a lower rate of synthesis of elongation factor Tu. Relaxed selection due to a lower protein synthetic load is also a plausible explanation for the accelerated rate of evolution of rrn16, but the available data are insufficient to test the hypothesis for this gene. The tufA and rrn16 genes in Polytoma oviforme, the sole member of a second nonphotosynthetic clade, are also functional but show no sign of relaxed selection.

Protozoa of a rotating biological contactor treatment plant in Mexico

A protozoological analysis of the biofilm developed on the discs of an RBC unit was performed using light microscopy; the species found belonged to 2 phyla: Sarcomastigophora with 23, and Ciliophora with 30 species. All isolates were free-living with the exception of Tritrichomonas fecalis. Most abundant species were: Euglena gracilis, Paramecium multimicronucleatum, Tetrahymena pyriformis, Polytoma uvella, Didinium nasutum, Bodo caudatus, Vorticella microstoma, Cyclidium glaucoma, Sathrophilus agitatus, Antophysa vegetans, and Urothrica farcta. Ciliates predominated over the flagellates in number and diversity. According to their locomotion most abundant species were free-swimming, followed by the attached, and crawling forms. In relation to their nutrition most frequent species were bacterivorous, saprozoic, and carnivorous. Values of the saprobic index calculated for each sampling station indicated that α-mesosaprobic conditions prevailed along the contactor with a zone of overlap with polysaprobic conditions at station IV. The RBC received an influent flow that varied from 1.5 to 13.5 L s−1 and a soluble BOD5 which range from 46 to 170 mg L−1. The contactor studied showed an overall BOD5 removal of 86% and a COD removal of 74%. DO levels influenced the values obtained for the saprobic index and the kind of protozoan populations and communities present. The biological findings (indicator species of the saprobic system), clearly reflected and agreed with the physicochemical results obtained simultaneously; the results showed that the system performed efficiently.

Cell walls of algae in the volvocales: Their sensitivity to a cell wall lytic enzyme and labeling with an anti-cell wall glycopeptide ofChlamydomonas reinhardtii

A cell wall lytic enzyme (gamete wall-autolysin) and a polyclonal antiserum raised against one of the major cell wall glycopeptides ofChlamydomonas reinhardtii were used to study their cross-reactivities with the cell walls of variety of members of the Volvocales. Lytic enzyme was able to digest completely the cell walls of five species ofChlamydomonas (C. reinhardtii group), six species ofGonium and two species ofAstrephomene. The colonial structures ofGonium andAstrephomene were broken into individual cells by exposure to the enzyme and protoplasts were then formed. These organisms also showed a strong cross-reactivity with anti-cell wall glycopeptide by an indirect-immunofluorescence test. The cell walls ofChlamydomonas angulosa, Dysmorphococcus globosus, Pandorina morum, Eudorina elegans, Volvulina steinii, Pleodorina california andVolvox carteri all showed a strong cross-reactivity to the antibody, although they were insensitive to the lytic enzyme. Many other species ofChlamydomonas, Carteria crucifera, Chlorogonium elongatum, Polytoma uvella, Haematococcus lacustris, Lobomonas piriformis, Phacotus lenticularis, Pteromonas angulosa, Stephanosphera pluvialis, andPyrobotrys casinoensis had cell walls which were resistant to the enzyme and showed no or weak cross-reactivity with the antibody. Based on the results, a possible evolutionary sequence from a unicellular relative ofC. reinhardtii to the multicellular algae is discussed.

Changes in sugar nucleotide pyrophosphorylases and starch during growth in cultures of Polytoma uvella and Polytomella papillota

Changes in starch, UDPG pyrophosphorylase, and ADPG pyrophosphorylase were followed during growth in cultures of two heterotrophic algae, Polytoma uvella and Polytomella papillata. There was a steady increase in the activities of the two sugar nucleotide pyrophosphorylases. The enzyme activities reached a maximum before starch production levelled off. At all stages of growth, UDPG pyrophosphorylase activity was much higher than ADPG pyrophosphorylase activity, in both algal cultures. Changes in UDPG and ADPG in cultures of Polytomella papillata showed correlation with changes in UDPG and ADPG pyrophosphorylase activities. Results suggest that the two pyrophosphorylases provide some of the starch precursor molecules.

Starch-producing Polytoma uvella lacks sucrose and sucrose-metabolizing enzymes

The absence of sucrose, sucrose synthetase and sucrose phosphate synthetase has been demonstrated by chromatographic, enzymatic and radioisotopic metho

Studies on the origin of amylose and amylopectin in starch granules. III. The effect of temperature on enzyme activities and amylose content

Starch composition (amylose content) and the activities of starch-synthesizing enzymes were determined in cultures of Polytoma uvella and potatoes kept at various temperatures. In general ADPG-α-glucan glucosyltransferases were much less thermostable in vivo than P- and Q-enzymes. Potato phosphorylase proved to be the most stable enzyme. In Polytoma both P/Q ratio and amylose content decreased at 30 °C, whereas they remained unchanged in potatoes grown at 30 °C. This confirms the results from earlier experiments which indicated that the P/Q ratio is correlated with amylose content, and is also in agreement with the simultaneous increase in amylose percentage and P/Q ratio observed during the development of starch granules in corn endosperm. The results are discussed in relation to the problem of amylose production in starch granules and the importance of phosphorylase in starch synthesis.

Changes in the free nucleotide pool during growth in cultures of Polytoma uvella.

The nucleotide components of Polytoma uvella grown in shaken cultures have been examined, and fluctuations in their individual concentrations assessed during the three phases of growth. The most marked change in the nucleotide pool occurs between 30–58 h (end of lag and onset of log phase), when a relatively large increase was observed in the concentration of all the components; the most significant increases were in the nucleoside 5′-triphosphates, UDP-glucose, and ADP-glucose. Before the end of log phase the concentrations of CTP, ATP, GTP, and UTP had decreased whereas those of UDP-glucose and ADP-glucose did not do so until after the onset of the stationary period. At this time the concentration of pyridine nucleotide had increased. There was a predominance of purine nucleotides throughout the growth of the cultures and further, at all stages, a preponderance of adenine and uracil nucleotides existed over those of cytosine and guanine. By 144 h (late stationary phase) the concentration of each nucleotide had fallen considerably.

Characterization of DNA components from some colorless algae

The DNA components of five colorless algae were characterized by their buoyant densities in cesium chloride. Two DNA components were detected in Polytoma obtusum and Polytoma uvella. Upon renaturation of the thermally denatured DNA the minor and approx. 15% of the major DNA component returned to their native densities. The buoyant densities of the major and minor DNA of P. obtusum and P. uvella are different from that of the morphologically and biochemically similar green alga Chlamydomonas reinhardtii. A major and a minor DNA component with the same buoyant densities as that of the green alga Euglena were also found in Astasia longa, which is morphologically similar to Euglena. The renaturation of the minor but not the major component was readily detectable by the change in buoyant density. Only one DNA component was detected in Polytomella agilis and Polytomella caeca. After thermal denaturation approx. 5% of each of these DNA components were renatured readily. Based on these data, the possible evolutionary origin of these colorless algae is discussed.

Studies on the Origin of Amylose and Amylopectin in Starch Granules. I. The Use of Polytoma uvella as a Source of NDPG‐Glucosyltransferases and some Properties of the Enzymes

AbstractThe alga Polytoma uvella was found to be a good source of both soluble and starch‐bound NDPG‐glucosyltransferase. The properties of the enzymes were similar to those of transferases from other plant sources, but differed in some respects. ADPG and UDPG acted as glucose donors, while maltose was inactive as glucose acceptor. Acetone preparations of the soluble enzyme were stable, but β‐amylase could not be completely removed without inactivating the transferase. They also contained oligosaccharides of the maltose series.

Studies on the Origin of Amylose and Amylopectin in Starch Granules. II. Changes in Amylose Content and Enzyme Activities in Cultures of Polytoma uvella

AbstractChanges in amylose percentage and the activities of starch‐synthesizing enzymes were followed in cultures of Polytoma uvella over a 6‐day period. Phosphorylase, ADPG‐glucosyltransferase, amylose content, ADPG and UDPG concentrations reached a peak in 96 hrs. old cultures. At this stage the cells of shaken cultures contained much higher enzyme activities and much more starch than those of stagnant cultures. After 96 hrs. phosphorylase and transferase activities declined sharply, amylose percentage decreased from 13.5 % to 6 %, but starch production continued, be it at a lower rate. Since Q‐enzyme activity increased during this period, it is suggested that the decrease in amylose percentage was due to preponderant production of amylopectin. A high correlation existed between amylose percentage and starchbound transferase. Experiments were initiated to bring about differential inactivation of phosphorylase and ADPG‐glucosyl‐transferase by growing cultures at higher temperatures.

The respiratory chain of colorless algae: III. Electron microscopy

Electron micrographs of KMnO4-fixed Prototheca zopfii, Polytoma uvella, and Polytomella agilis show the presence of mitochondria with typical discoidal cristae of random orientation, a result expected from their cytochrome content. The mitochondria are usually localized near the outer margin of the cell. All three organisms contain starch granules and proplastid-like structures, both bounded by two membranes. Although Prototheca has been called “a colorless Chlorella,” the existence of significant differences in their ultrastructure makes this classification unlikely. Astasia longa does not contain cytochrome oxidase, but its mitochondria show the same type of ultrastructure as the Chlorophyta, and it too has a eucaryotic cellular structure. The Cyanophyta, Leucothrix mucor, and Vitreoscilla sp. have a typical procaryotic cellular structure, a result consistent with the fact that their respiratory characteristics are like those of the Eubacteria. The cell walls of Leucothrix and Vitreoscilla resemble the cell walls of their procaryotic relatives, the photosynthetic blue-green algae.

単細胞藻類からのジメチルサルファィドの生産

Volatile sulfur compounds evolved from various unicellular algae during cultivation were fractionated to three different parts: thioether fraction, mercaptan fraction and H2S fraction. Major component of the volatile sulfur compounds produced from Amphidinium carterii, Glenodinium sp., Chlamydomonas sp., Nanochloris oculata, Haematococcus pluvialis, Polytoma uvella, Chlamydomonas komma, Scenedesmus obliquus, Chlorella vulgaris and Chlorella pyrenoidosa was H2S, and that from Astasia longa and Euglena gracilis was mercaptan. Gyrodinium cohnii, a heterotrophic dinoflagellate of marine origin evolved thioether as the major product. The thioether fraction obtained from Gyrodinium cohnii, was found by gas chromatographic analysis to be principally composed of dimethyl sulfide. These data suggested that the ability of unicellular algae to produce thioether, mercaptan or H2S was related to some extent to the metabolic type or the environmental factors of natural habitats of the organism. This work was supported in part by research grant from the Ministry of Education, Japan.

The Sterol Content of Some Protozoa

SYNOPSIS. The sterols of a number of protozoa grown in axenic culture have been examined. Ergosterol is present in Polytoma uvella, Astasia ocellata, Haematococcus pluvialis, Crithidia oncopelti, Prototheca zopfii, Chilomonas paramoecium and Trypanosoma mega, all grown on sterol‐free media. Ergosterol is also present in the culture form of Trypanosoma rhodesiense and occurs together with cholesterol and an unidentified sterol in Peranema trichophorum grown in the presence of cholesterol. P. uvella, A. ocellata, H. pluvialis and C. oncopelti also contain a sterol which is either spinasterol or its isomer chondrillasterol. The main sterol of Ochromonas malhamensis is probably poriferasterol, which is also present in C. paramoecium. Cholesterol, probably from the environment, is the only sterol in the blood form of T. rhodesiense. Tetrahymena pyriformis and Hartmannella rhysodes do not synthesize sterols.

Studies on the metabolism of the protozoa. The molecular structure of a starch-type polysaccharide from Polytoma uvella.

1. Polytoma uvella, when grown in an acetate-containing medium, synthesizes a starch-type polysaccharide. 2. The starch differs from normal plant starches in having a lower iodine affinity, and in being relatively insoluble; this latter property makes fractionation difficult. 3. The starch contains about 16% of amylose, and on fractionation yields a branched amylopectin component that is similar in structure to a typical plant starch amylopectin.

Some growth experiments withPolytoma uvella on synthetic media

1) Polytoma uvella (strain 1) grows excellently on the following basic medium: sodium acetate, 0.2%; asparagine, 0.2%; K2HPO4, 0.02%; MgSO4.7H2O, 0.01%; Na2CO3, 0.02%; pH=7.5 (in distilled water). Caramel, CaCl2, FeCl3 or other additional trace metals are not required. 2) Replacement of asparagine by glycine resulted in very poor growth. Addition of thiamin, vitamin B12 or trace elements did not improve the glycine medium. It is suggested thatPolytoma uvella needs small amounts of a Krebs cycle intermediate for good growth.

Movement and nucleoside polyphosphatase activity of isolated flagella from Polytoma uvella

Flagella can be quantitatively removed and collected from Polytoma uvella by suspension in 50–60 per cent glycerol at subzero temperatures. When suspended in solutions containing ATP, these isolated flagella beat vigorously and swim forward through the medium at up to a sixth their normal speed. The flagella continue to dephosphorylate ATP at the rate required to supply energy for normal movement, although less than 5 per cent of this energy is used in moving against the viscous resistance of the medium. The rate of flagellar beating depends on the ATP concentration, but the rate of dephosphorylation is nearly constant above 2 × 10 −5 M. Although other nucleoside triphosphates are dephosphorylated at up to a quarter the rate observed with ATP, they do not induce movement, nor do the corresponding diphosphates. ADP induces movement and is dephosphorylated at the same rate as ATP because the flagella possess an adenylate kinase. The high specificity of the adenylate kinase suggests that it might play a role in the relaxation portion of the cycle of flagellar contraction.

Acetylcholinesterase in flagellated systems

1. 1. Cholinesterase could not be detected in Polytoma uvella, Polytomella caeca, or Tetrahymena pyriformis W homogenates. 2. 2. Trout sperm and perch sperm possessed activity corresponding to the splitting of 0.02 to 0.14 μg/h/mg at 37°. This activity was specific for acetylcholine. 3. 3. The enzyme of perch sperm was found to be located in the head of the spermatozoa.