Discovery of the first virus, the tobacco mosaic virus: 1892 or 1898?

Two scientists contributed to the discovery of the first virus, Tobacco mosaic virus. Ivanoski reported in 1892 that extracts from infected leaves were still infectious after filtration through a Chamberland filter-candle. Bacteria are retained by such filters, a new world was discovered: filterable pathogens. However, Ivanovski probably did not grasp the full meaning of his discovery. Beijerinck, in 1898, was the first to call 'virus', the incitant of the tobacco mosaic. He showed that the incitant was able to migrate in an agar gel, therefore being an infectious soluble agent, or a 'contagium vivum fluidum' and definitively not a 'contagium fixum' as would be a bacteria. Ivanovski and Beijerinck brought unequal but decisive and complementary contributions to the discovery of viruses. Since then, discoveries made on Tobacco mosaic virus have stood out as milestones of virology history.     

Beijerinck's work on tobacco mosaic virus: historical context and legacy

Beijerinck's entirely new concept, launched in 1898, of a filterable contagium vivum fluidum which multiplied in close association with the host's metabolism and was distributed in phloem vessels together with plant nutrients, did not match the then prevailing bacteriological germ theory. At the time, tools and concepts to handle such a new kind of agent (the viruses) were non-existent. Beijerinck's novel idea, therefore, did not revolutionize biological science or immediately alter human understanding of contagious diseases. That is how bacteriological dogma persisted, as voiced by Loeffler and Frosch when showing the filterability of an animal virus (1898), and especially by Ivanovsky who had already in 1892 detected filterability of the agent of tobacco mosaic but kept looking for a microbe and finally (1903) claimed its multiplication in an artificial medium. The dogma was also strongly advocated by Roux in 1903 when writing the first review on viruses, which he named 'so-called "invisible" microbes', unwittingly including the agent of bovine pleuropneumonia, only much later proved to be caused by a mycoplasma. In 1904, Baur was the first to advocate strongly the chemical view of viruses. But uncertainty about the true nature of viruses, with their similarities to enzymes and genes, continued until the 1930s when at long last tobacco mosaic virus particles were isolated as an enzyme-like protein (1935), soon to be better characterized as a nucleoprotein (1937). Physicochemical virus studies were a key element in triggering molecular biology which was to provide further means to reveal the true nature of viruses 'at the threshold of life'. Beijerinck's 1898 vision was not appreciated or verified during his lifetime. But Beijerinck already had a clear notion of the mechanism behind the phenomena he observed. Developments in virology and molecular biology since 1935 indicate how close Beijerinck (and even Mayer, Beijerinck's predecessor in research on tobacco mosaic) had been to the mark. The history of research on tobacco mosaic and the commitments of Mayer, Beijerinck and others demonstrate that progress in science is not only a matter of mere technology but of philosophy as well. Raemaekers' Mayer cartoon, inspired by Beijerinck, artistically represents the crucial question about the reliability of our images of reality, and about the scope of our technological interference with nature.     

Historical microbiology: revival and phylogenetic analysis of the luminous bacterial cultures of M. W. Beijerinck

Luminous bacteria isolated by Martinus W. Beijerinck were sealed in glass ampoules in 1924 and 1925 and stored under the names Photobacterium phosphoreum and 'Photobacterium splendidum'. To determine if the stored cultures were viable and to assess their evolutionary relationship with currently recognized bacteria, portions of the ampoule contents were inoculated into culture medium. Growth and luminescence were evident after 13 days of incubation, indicating the presence of viable cells after more than 80 years of storage. The Beijerinck strains are apparently the oldest bacterial cultures to be revived from storage. Multi-locus sequence analysis, based on the 16S rRNA, gapA, gyrB, pyrH, recA, luxA, and luxB genes, revealed that the Beijerinck strains are distant from the type strains of P. phosphoreum, ATCC 11040(T), and Vibrio splendidus, ATCC 33125(T), and instead form an evolutionarily distinct clade of Vibrio. Newly isolated strains from coastal seawater in Norway, France, Uruguay, Mexico, and Japan grouped with the Beijerinck strains, indicating a global distribution for this new clade, designated as the beijerinckii clade. Strains of the beijerinckii clade exhibited little sequence variation for the seven genes and approximately 6300 nucleotides examined despite the geographic distances and the more than 80 years separating their isolation. Gram-negative bacteria therefore can survive for many decades in liquid storage, and in nature, they do not necessarily diverge rapidly over time.     

Novel principles in the microbial conversion of nitrogen compounds

Some aspects of inorganic nitrogen conversion by microorganisms like N2O emission and hydroxylamine metabolism studied by Beijerinck and Kluyver, founders of the Delft School of Microbiology, are still actual today. In the Kluyver Laboratory for Biotechnology, microbial conversion of nitrogen compounds is still a central research theme. In recent years a range of new microbial processes and process technological applications have been studied. This paper gives a review of these developments including, aerobic denitrification, anaerobic ammonium oxidation, heterotrophic nitrification, and formation of intermediates (NO2-, NO, N2O), as well as the way these processes are controlled at the genetic and enzyme level.     

Nitrate Reductase and Chlorate Toxicity in Chlorella vulgaris Beijerinck

A study of the growth-inhibiting effect of chlorate on the Berlin strain of Chlorella vulgaris Beijerinck provided complete confirmation of the theory of chlorate toxicity first proposed by Åberg in 1947. Chlorate was toxic to the cells growing on nitrate, and relatively nontoxic to the cells growing on ammonium. The latter cells contained only 0.01 as much NADH-nitrate reductase as the nitrate-grown cells. Chlorate could substitute for nitrate as a substrate of the purified nitrate reductase with Km = 1.2 mm, and V_max = 0.9V_max for nitrate. Bromate, and to a much smaller extent, iodate, also served as alternate substrates. Nitrate is a reversible competitive inhibitor of chlorate reduction, which accounts for the partial reversal, by high nitrate concentrations, of the observed inhibition of cell growth by chlorate. During the reduction of chlorate by NADH in the presence of purified nitrate reductase, there was a progressive, irreversible inhibition of the enzyme activity, presumably brought about by the reduction product, chlorite. Both the NADH-nitrate reductase activity and the associated NADH-cytochrome c reductase activity were inactivated to the same extent by added chlorite. The spectral properties of the cytochrome b₅₅₇ associated with the purified enzyme were not affected by chlorite. The inactivation of the nitrate reductase by chlorite could account for the toxicity of chlorate to cells grown on nitrate, though the destruction of other cell components by chlorite or its decomposition products cannot be excluded.     

Activation of nitrate reductase by oxidation

The activation of the inactive form of the nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) present in cell-free extracts of Chlorella vulgaris Beijerinck requires an oxidizing agent. Ferricyanide causes conversion of the proenzyme to active enzyme within a few minutes, even at 0°C. Molecular oxygen causes a slow activation which requires many hours at room temperature, and never reaches the high activity level attained with ferricyanide. In unfractionated extracts, CO inhibits the activation by molecular O₂. The sensitivity of this activation to CO may account for the in vivo sensitivity of nitrate reduction to CO in these algae.     

Synthesis of Nitrate Reductase in Chlorella: II. EVIDENCE FOR SYNTHESIS IN AMMONIA-GROWN CELLS

Antiserum was prepared against nitrate reductase (EC 1.6.6.1) purified to homogeneity from Chlorella vulgaris Beijerinck. Both crude antiserum and anti-nitrate reductase antibodies prepared from it were used as re-agents to study the synthesis of nitrate reductase. Cell extracts from cultures which were grown with ammonia salts as the sole source of nitrogen contained almost no active enzyme. These extracts did contain material which binds to antibody and is thus immunologically related to purified nitrate reductase. The presence of this cross reacting material in cell extracts was detected by the ability of these extracts to (a) lower the titer of antisera; (b) form a biphasic precipitin curve with purified antibody; and (c) increase the peak height of a standard amount of purified nitrate reductase in rocket immunoelectrophoresis assay. These results suggest that ammonia-grown cells contain nitrate reductase precursor protein.     

100 years of virology: from vitalism via molecular biology to genetic engineering

The identification of the causative agent of tobacco mosaic disease as a novel pathogen by the Dutch microbiologist Beijerinck is now acknowledged as being the foundation of virology as a discipline distinct from bacteriology. However, as this was contrary to the prevailing theories of the time, it took many years for virology to become firmly established as a separate discipline. The history of virology illustrates how accepted concepts in science evolve by trial and error and the need for a balanced approach when manipulating natural systems.     

Influence of traumatic acid on growth and metabolism of Chlorella vulgaris under conditions of salt stress

The aim of this work was to study the role of traumatic acid (TA) in the adaptation of green algae Chlorella vulgaris Beijerinck to salt stress. In order to achieve this aim, studies were conducted concerning the influence of exogenous TA at a concentration of 10^?5 M combined with sodium chloride (NaCl) at various concentrations (10^?7–10^?4 M), on the growth of C. vulgaris and the levels of basic biochemical parameters, such as chlorophylls a and b, monosaccharides, cell proteins and the activity of superoxide dismutase enzyme. The studies carried out demonstrated that TA eliminated the negative effects of NaCl at all test concentrations and also increased the content of carotenoids, monosaccharides and cell proteins.     

THE HOST RANGE OF THE CHLORELLAVOROUS BACTERIUM ("VAMPIROVIBRIO CHLORELLVORUS")

The chlorellavorous bacterium . "Vampirovibrio chlorellavorus" Gromov et Mamkaeva, grows only by killing and consuming the cell contents of species of the green alga Chlorella Beijerinck. Of the 76 algal strains examined in this study, the bacterium attacks all 31 strains of the species C. vulgaris. C. sorokiniana, and C. kessleri, but attacks only two of 39 strains of nine other Chlorella species. Neither of two Prototheca strains was susceptible to attack. This narrow host specificity may be related to cell surface properties .     

The origin of modern plant virology

Plant virology, born with Mayer's work, saw a first (embryonic) phase of development during two decades (1900-1920) with outstanding contributions from Dimitri Ivanovski, Martinus Beijerinck, Erwin Baur and Harry Allard. Between 1920 and 1930 a second phase saw the elaboration of surprising hypotheses concerning the enigmatic nature of viruses and experimental evidence of great stress was obtained. Revolutionary renewal began from the mid-1930s on the basis of a body of knowledge which was organically assembled into the first textbook of plant virology published by Kenneth Smith in 1933. In 1922, the geneticist Hermann Muller put forward the hypothesis that considered viruses as possible genes. The theory was resumed in an apparently independent way by Benjamin Duggar and Joanne Karrer Armstrong in 1923, who considered TMV a biocolloidal self-reproducing protein, like genes appeared to be. This hypothesis, even if neglected by virologists, anticipated by some decades the functional nature of viruses and represented the first conceptual response to virus enigma. Considerable experimental results were obtained by James Johnson, who showed that plants could be infected by different viruses and who introduced a first rational system of plant virus classification. Harold McKinney showed that TMV could mutate. Harold Storey, Kenneth Smith and Harry Severin demonstrated that several viruses could be transmitted by insects and supplied the first interpretation of the relationship between virus and insect. Mayme Dvorak and Helen Purdy obtained the first experimental evidence of the antigenic power of plant viruses. Virus purification, first tentatively accomplished with physical methods, was brilliantly performed by chemical means. Finally, Francis Holmes elaborated the first suitable test to estimate virus infectivity. The evolution of plant virology from an empirical discipline to a biological science took place thanks to the work of one group of American and English scientists who must be regarded as the fathers of modern plant virology.     

Measurement of enzymes related to sucrose metabolism in permeabilized Chlorella vulgaris cells

Sucrose phosphate synthase (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14), sucrose synthase (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were measured in toluene permeabilized cells of Chlorella vulgaris Beijerinck. All three activities were detected at all stages of the growth curve; sucrose synthase and sucrose phosphate synthase showed a zone of maximum activity, while invertase increased with time of growth. Sucrose phosphate synthase and sucrose synthase (sucrose synthesis direction) were stimulated by divalent cations and inhibited by UDP. This inhibition could be reversed by Mg²⁺ or Mn²⁺. Sucrose phosphate synthase activity was inhibited by inorganic phosphate and was enhanced by glucose-6-phosphate, but was insensitive to sucrose. Arbutine decreased sucrose synthase activity in both directions. Sucrose cleavage was inhibited by divalent cations and by pyrophosphate. The effects on the enzyme activities of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid, abscisic acid and kinetin in the growth medium were investigated. Sucrose synthase activity was practically unaffected by all plant hormones tested, except for the presence of kinetin which stimulated the activity. Sucrose phosphate synthase activity was increased by both kinetin and abscisic acid. The effect of the latter was partially reversed by the presence of gibberellic acid. 2,4-D and kinetin were potent stimulators of invertase activity.     

DISTINCTION BETWEEN MULTIPLE ISOLATES OF CHLORELLA VULGARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE) AND TESTING FOR CONSPECIFICITY USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM AND ITS RDNA SEQUENCES1

Multiple strains of individual algal species are available from public culture collections, often with the same isolate being maintained in parallel at a number of collections under different culture regimes. To unravel genomic variation and to identify unique genotypes among such multiple strains, two approaches were used on a sample of 29 strains of Chlorella vulgaris Beijerinck, an alga of great value for applied research, from five culture collections. With the exception of two strains, internal transcribed spacer rDNA sequence data substantiated conspecificity of the studied strains and only minor sequence differences with the authentic “Beijerinck isolate” were observed. Amplified fragment length polymorphism (AFLP) detected considerable genomic variation when rDNA sequences were identical. Band detection and the construction of a binary matrix from AFLP patterns for phylogenetic analyses were fully automated, but comparison of similar patterns still required manual refinement. The AFLPs distinguished 11 unique genotypes and provided robust support for the presence of five cryptic species. This finding advocates the need to carefully record which strain has been used in any experiment or in applied research, because genomic variation may also correspond to differences in physiological/biochemical properties. No genomic differences could be detected between duplicate strains of the same isolate that were maintained by continuous subculturing over many decades or within those stored at ultralow temperatures.     

BIOCHEMICAL TAXONOMY AND MOLECULAR PHYLOGENY OF THE GENUS CHLORELLA SENSU LATO (CHLOROPHYTA)

A multimethod approach was used to characterize unicellular green algae that were traditionally assigned to the genus Chlorella Beijerinck and to resolve their phylogenetic relationships within the Chlorophyta. Biochemical, physiological, and ultrastructural characters, together with molecular data such as DNA base composition and DNA hybridization values, were compared with a molecular phylogeny based on complete 18S rRNA sequences. Our results show that Chlorella taxa are dispersed over two classes of chlorophytes, the Trebouxiophyceae and the Chlorophyceae. We propose that only four species should be kept in the genus Chlorella (Chlorophyta, Trebouxiophyceae): C. vulgaris Beijerinck, C. lobophora Andreyeva, C. sorokiniana Shih. et Krauss, and C. kessleri Fott et Nováková. Common characteristics of these taxa are glucosamine as a dominant cell wall component and the presence of a double thylakoid bisecting the pyrenoid matrix. Norspermine, norspermidine, and secondary carotenoids are never produced. Other "Chlorella" species belong to different taxa within the Trebouxiophyceae ( "C." protothecoides = Auxenochlorella protothecoides [Krüger] Kalina et Punčochářová, "C." ellipsoidea, "C." mirabilis, "C." saccharophila, and "C." luteoviridis ) and Chlorophyceae ( "C." zofingiensis and "C." homosphaera = Mychonastes homosphaera Kalina et Punčochářová). The latter taxa can easily be recognized by the production of secondary carotenoids under nitrogen-deficient conditions.     

A study of the budding ofSaccharomyces uvarum Beijerinck with the scanning electron microscope

Vegetative cells ofSaccharomyces uvarum Beijerinck in the exponential growth phase were examined with the scanning electron microscope. The existence of two types of scars — birth scars and bud scars — was confirmed. Birth scars had larger diameters than bud scars; both remained visible on old cells. The distribution of the buds on the mother cell did not appear to be a random one: there seemed to be a more or less emphasized cell polarity. The author wishes to thank Mr. Bert for technical assistance in the use of the scanning electron microscope.     

Photosynthetic Apparatus Formation during the Cell Cycle of Chlorella

Synchronous cell division in cultures of Chlorella vulgaris Beijerinck was induced by intermittent illumination: 9 hours light, 6 hours darkness. The rate of photosynthetic O₂ evolution per cell increases 4-fold in a one-step manner at the beginning of the light period, to the same extent as the increase in cell number. Over the division cycle, the following accumulation times during the light period were found: chlorophyll a, between 2 and 8 hours, chlorophyll b, between 5 and 8 hours, reaction centers of photosystems I and II, between 2 and 6 hours; and cytochrome f, between 2.5 and 5 hours. Cytochrome f accumulation is closely followed by an increase in amplitude of the rapid phase in light-induced absorption increase at 520 nanometers and in intensity of the delayed light emission. Enhancement of the delayed fluorescence yield per flash under continuous illumination (caused by the establishment of the pH difference across the thylakoid membrane) is maximal by the first hour of the light period.     

Molecular characterization of Chlorella cultures of the National Institute for Environmental Studies culture collection with description of Micractinium inermum sp. nov., Didymogenes sphaerica sp. nov., and Didymogenes soliella sp. nov. (Chlorellaceae,Trebouxiophyceae)

Chlorella Beijerinck (Chlorellaceae, Trebouxiophyceae) strains from the collection of the National Institute for Environmental Studies (NIES) were characterized using gene sequence data. The misidentification of a number of strains was rectified. Chlorella vulgaris Beijerinck NIES‐2173 was reclassified as C. sorokiniana Shihira et Krauss. Chlorella sp. NIES‐2171 was described as a new species in the genus Micractinium Fresinius, M. inermum Hoshina et Fujiwara. Chlorella sorokiniana NIES‐2167 and Chlorella sp. NIES‐2330 were found to be phylogenetically related to Didymogenes Schmidle. We propose these two strains be transferred to the genus Didymogenes and given new names: D. sphaerica Hoshina et Fujiwara and D. soliella Hoshina et Fujiwara. Taxonomic decisions were primarily based on small subunit‐internal transcribed spacer ribosomal DNA phylogeny for genus assignment and ITS2 sequence‐structure to determine species autonomy. Our findings suggest that this strategy is the most effective way to use the species concept among autosporic coccoids.     

Physiologische Untersuchungen zur Bioluminescenz von Vibrio luminosus Beijerinck

Zusammenfassung Es wird eine Methode zur Anzucht von Vibrio luminosus Beijerinck und zur fortlaufenden Messung der Lichtintensität mit Sekundärelektronenvervielfachern beschrieben. Vergleichende Licht-und Atmungsmessungen ergeben, daß die Lichtemission erst nach Erreichen des Atmungsmaximum ansteigt. An Hand der Luminescenzbeeinflussung durch industrielle Schadstoffe wird gezeigt, daß Leuchtbakterien für entsprechende Biotests herangezogen werden können. Voraussetzung dafür ist die Kenntnis des Stoffwechsels und der optimalen Kulturbedingungen. Auf die Temperatur- und pH-Abhängigkeit der Lichtemission und die spektrale Verteilung des emittierten Lichtes wird eingegangen.

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Physiological studies in bioluminescence of Vibrio luminosus Beijerinck

Summary A method is established for the cultivation of Vibrio luminosus Beijerinck and continuous registration of light intensity with photomultipliers. Comparing examinations of luminescence and respiration demonstrate that light emission is increasing only after the maximum of respiration has been reached. The intensity of bioluminescence is influenced by noxious substances of industrial origin and luminous bacteria can be used as test organisms. The general physiological characteristics of the test strain and the foundations of cultivation must be known for biotests. The effect of temperature and pH on the light emission and the spectral distribution of the emitted light is shown.

     

Cyanide formation in preparations from Chlorella vulgaris Beijerinck: Effect of sonication and amygdalin addition

Summary A critical evaluation of a method for recovering HCN from cell extracts is presented. Since crude extracts often bind or metabolize HCN extensively, the HCN recovered by distillation at room temperature represents only the difference between production and consumption. Sonication leads to HCN release from the alga, Chlorella vulgaris Beijerinck. Illumination of extracts at high light intensity in oxygen, with added Mn²⁺ and peroxidase, also stimulates HCN production. In both processes, the HCN is probably formed by oxidation of nitrogenous precursors. Chlorella extracts cause formation of HCN from added amygdalin. No evidence was found, however, for the presence of cyanogenic glycosides in the algae.     

Screening,Identification and Distribution of Endophytic Associative Diazotrophs Isolated from Rice Plants

29 strains of endophyte associated diazotrophs isolated from rice ( Oryza sutiva L. ) "Yuefu" plant were selected by in vitro acetylene reduced activity and 15 N2-flxing activity determination. They were identificated into 14 species of 9 genera: Agrobacterium tumefaciens (Smith et Townsend) Conn, A. radiobacter (Beijerinck et van Delden) Conn; Alcaligenes piechaudii Kiredjian et al., Al. denitrificans (Leifson et Hagh) Ruger et Tan; Bacillus sphaericus Meyer et Neide, B. licheniforrnis Weigmann Chester, B. cereus Frankland et Frankland; Chryseomonas luteola (Kodama, Kinnura et Komagata) Holmes et al.; Enterobacter cloacae ( Jordan ) Honnaeche et Edwards, E. sakazakii Famer et al.; E. agglomeraus (Beijerinck) Ewing et Fife; Pseudomonas pseudoalcaligenes Stanier, P. alcaligeaes Monias, P. putida Biotype A Evans and Aeromonas Kluyver et van Niel, Serratia Bizio, Staphyloccocus Rosenbach, Xanthomonas Dowson. Among them C. luteola, E. sakazakii, E. agglomeraus, P. pseudoalcaligenes have not been previously reported as diazotrophs. Studying on the distribution of endophytic dizoatrophs in rice seeds and rice plants has demonstrated that the diversity of endophytic associated diazotrophs in roots was more than that in other organs of rice plant. Endophytic associative diazotroph E. cloacae not only occurred on the surface but also occurred intercellularly in R48 rice roots as observed with scanning electron microscopy and transmission microscopy.