STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM: I. OXIDATION OF SULFUR

1. Thiobacillus thiooxidans, isolated in our laboratory, was foundto oxidize sulfur, but not thiosulfate. Tetrathionate is alsooxidized slightly. Its ability to oxidize sulfur is inactivatedeven by such a mild treatment as keeping the cells in a frozenstate.
2. Inhibitory action of alcohols on the sulfur oxidationincreasesas the length of carbon chain of alcohols increases.Carboxylicacids do not inhibit the sulfur oxidation at pH abovetheirpK, while they strongly inhibit the reaction at pH belowthepK.
3. The sulfur oxidation is inhibited by cyanide, azide,diethyldithiocarbamateand carbon monoxide, and the inhibitionby carbon monoxide isnot reversed by light. These results suggestthe presence ofmetal enzymes in the sulfur oxidation system.The terminal enzymeof this reaction appears to be differentfrom the usual cytochromeoxidase.

(Received May 13, 1960; )     

Studies on the metabolism of a sulfur-oxidizing bacterium VI1. Fractionation and reconstitution of the elementary sulfur-oxidizing system of Thiobacillus thiooxidans

The sulfur-oxidizing system of a strain of Thiobacillus thiooxidanswas obtained in cell-free state. The system is resolved intothree fractions and can be reconstituted from these fractions.Both the soluble and particulate fractions are required forthe oxidation of elementary sulfur. The soluble fraction wasfurther separated into two fractions, the collodion membrane-permeable(S-P)and the impermeable(S-IP). S-P contains a low molecular weight,relatively heat stable substance(s) which is indispensable forthe reconstitution of the sulfur-oxidizing system and was identifiedas a pyridine nucleotide. The function of S-P can be replacedby NAD or NADP, but not by cysteine nor GSH. Oxidation of NADH₂ and NADPH₂ is catalyzed by the particulatefraction. Oxidation of the latter is much more rapid than thatof the former. Oxidation of NADPH₂ as well as sulfur oxidationis inhibited by cyanide, pCMB and CO, the CO-inhibition beingphoto-irreversible. However, strong inhibitors of sulfur oxidationsuch as DDC, 8-hydroxyquinoline and salicylaldoxime have noeffect on the oxidation of NADPH₂. The optimum pH values for sulfur and sulfite oxidations by thecell-free extract are shifted to the neutral side in comparisonwith pH values by intact cells. ¹V = References(I). ²Partly supported by a grant from the Ministry of Education. (Received April 3, 1969; )     

Pigment Composition in the Reaction Center Complex from the Thermophilic Green Sulfur Bacterium, Chlorobium tepidum: Carotenoid Glucoside Esters, Menaquinone and Chlorophylls

Distribution of pigments in the reaction center (RC) complex,chlorosomes and chlorosome-free membranes prepared from thegreen sulfur bacterium, Chlorobium tepidum, was analyzed. TheRC complex contained approximately 40 molecules of bacteriochlorophyll(BChl) a per P840, half of which are estimated to be in theFenna-Matthews-Olson (FMO) protein. Carotenes (2 molecules perP840) occupied only one third of the total carotenoids. Theremaining carotenoids (4 to 5 molecules per P840) were OH-chlorobacteneglucoside ester and OH-     

Sulfur requirement of Gonium (Volvocaceae)

The sulfur requirement of six strains of three species of Goniumhas been investigated. These strains can grow well with sulfide,sulfite, bisulfite, thiosulfate or sulfate in light and darkness.They are the first algae shown to utilize sulfide as a sulfursource. However, organic sulfur sources (methionine, cystine,cysteine, homocysteine, homocystine and taurine) were ineffectivefor growth of Gonium. (Received December 6, 1975; )     

Studies on Nucleolus and Nucleolar Chromosomes in Angiosperms. XI. Plantago L.

The number and morphology of nucleotar chromosomes is describedfor 11 species of Plantago. The possible significance of interspecificdifferences in number and morphology and their potential valuein phylogenetic studies are discussed. Plantaginaceae, Plantago L., karyology, NOR, nucleolus, nucleolar bivalents, NOR evolution     

Studies on the metabolism of a sulfur-oxidizing bacterium V. Comparative studies on sulfur and sulfite oxidizing systems of Thiobacillus thiooxidans

Properties of the oxidation systems of sulfur and sulfite ofa sulfur oxidizing bacterium, Thiobacillus thiooxidans, arecompared by using various inhibitors. Oxidation of sulfur isinhibited by a low concentration of monoiodoacetic acid, NEMand pCMB. Inhibition by pCMB is diminished by the addition ofan equivalent amount of cysteine to that of added pCMB. Althoughinhibition by pCMB is also observed in the oxidation of sulfite,it is not diminished by the addition of excess cysteine andthe extent of inhibition is lower than that in the oxidationof sulfur. Metal chelating agents, such as DDC, 8-hydroxyquinoline, salicylaldoximeand neocuproine have inhibitory effects on the oxidation ofsulfur but do not affect the oxidation of sulfite. Carbon monoxide inhibits the oxidation of sulfur photo-irreversiblyand the oxidation of sulfite photo-reversibly. Alcohols and organic acids, inhibit the oxidation of both sulfurand sulfite. The cell-free extract prepared by sonic disruptionof cells can oxidize sulfite, but not sulfur. The sulfur oxidizingextract can be, however, prepared by disruption under a nitrogenatmosphere. Both the soluble and participate fractions are requiredfor the oxidation of sulfur, while sulfite oxidation is catalyzedby the participate fraction alone. ¹Partly supported by a grant from the Ministry of Education.     

ROLE OF SULFUR IN THE CELL DIVISION OF CHLORELLA, WITH SPECIAL REFERENCE TO THE SULFUR COMPOUNDS APPEARING DURING THE PROCESS OF CELL DIVISION. I.

1. The role of sulfur in the cell division of Chlorella was studiedby following the fate of the sulfur supplied to the sulfur-deficientcells using ³⁵S as a tracer.
2. The sulfur-deficient cells whichwere unable to perform celldivision were made capable of divisionby the provision of ³⁶S-labeledsulfate under non-photosynthesizingconditions. Soon after theprovision of sulfate the labeledsulfur went rapidly into thecold perchloric acid (PCA)-solublefraction of algal cells,almost entirely in the form of sulfateand/or some other inorganicsulfur substance (s). With the lapseof time, more or less remarkablechanges occurred in the patternof ³⁵S-distribution in differentfractions of cell material.It was noticed that, at the onsetof cell division, a sulfur-containingpeptide-nucleotide compound(s)(SPN), which has been reportedearlier, appeared in a largequantity in the cold PCA-solublefraction, and that its quantitydecreased gradually during thesubsequent process of cell division,suggesting that the compoundwas transformed into some othersubstance (s), presumably withits nucleotide moiety going intonucleic acids and the peptidemoiety going into some essentialproteins.
3. Another noteworthyphenomenon observed during the process ofcell division wasthe incorporation of ³⁶S in a group of hotPCA-soluble substances.These sulfur substances were revealedto be sulfur-containingnucleotidic compounds, which might possiblybe some essentialcomponents of, or substances in close relationto, deoxypentosenucleic acid (DNA).

(Received March 1, 1960; )     

Types, Formation, and Metabolism of Cytokinins in Leaves of Alnus Glutinosa (L.) Gaertn.

The nature of the cytokinins present in mature, expanded leavesof alder (Alnus glutinosa (L.) Gaertn.) was investigated usinga chromatographic system capable of partially resolving zeatinfrom (?)-dihydrozeatin. A zeatin-like cytokinin, present asboth a ‘free’ form and as a polar conjugate, postulatedto the the O-ß-D-glucoside of zeatin, accounted forthe bulk of the cytokinin activity (detected by the soybeancallus assay). Studies on the fate of [8-¹⁴C]zeatin supplied to detached alderleaves indicated this cytokinin to undergo extensive metabolism.An appreciable proportion of the radioactivity was incorporatedinto 80% methanol-insoluble and soluble acidic/neutral fractions,while adenosine- and adenine-like peaks were prominent metabolitesin the basic n-butanol-soluble fraction. Small amounts of glucosides,with the properties of both zeatin-O-ß-D-glucosideand dihydrozeatin-O-ß-D-glucoside were formed, thelatter becoming the most prominent with time. The ability of alder leaves to form glucoside directly from(?)-dihydrozeatin was assessed using the soybean callus assay.(?)-Dihydrozeatin was subject to a relatively rapid, continuousand substantial conversion to its putative O-ß-D-glucosideand cytokinin activity was consequently conserved while, incontrast, leaves supplied with zeatin exhibited a progressiveloss of cytokinin activity and, in agreement with the radioactivityexperiments, produced only a small amount of activity attributableto the putative O-glucosides. The significance of the observed cytokinin metabolism is discussedin relation to the endogenous cytokinin status of the leaf.     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM III. POSTOXIDATIVE FIXATION OF CARBON DIOXIDE

1. The cells of Thiobacillus thiooxidans, which had been in contactwith sulfur or sulfide in air (or CO₂-free air), could fix addedCOa very rapidly after replacing air with nitrogen. This fixationis designated as the postoxidative fixation.
2. "Preoxidation"of the sulfur compounds is mandatory for theoccurrence of thepostoxidative fixation.
3. The cells which had preliminarilyoxidized sulfide could notshow the CO₂-fixation, when theywere placed under an anaerobiccondition in the absence of thesulfur compound.
4. These results indicate that sulfur compoundsmay have an importantrole as the electron donor for the reductionof CO₂, besidestheir role as the substrate of respiration tosecure energyfor the fixation of CO₂

(Received March 6, 1962; )     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM II. THE SYSTEM OF CO2-FIXATION IN THIOBAC1LLUS THIOOXIDANS

1. In the early stage of CO₂-fixation by Thiobacillus thiooxidans,which was incubated aerobically in the presence of sulfur, mostpart of the fixed carbon was found in the phosphate ester fraction.
2. The fixation was inhibited by NaF, picolinic acid, PCMB, azide,dipyridyl, o-phenanthroline, monoiodoacetic acid, and arsenite,each in the concentration range where the sulfur oxidation wasnot affected strongly.
3. The crude extract of this organismcould fix CO₂ in the presenceof ATP, R-5-P and Mg⁺⁺. Most partof the fixed carbon was foundin PGA.
4. The crude extract showedthe CO₂-fixation coupled with the H₂S-oxidationin the presenceof ADP.
5. An appreciable reduction of PGA could not be detectedin thepresence of reducing systems, involving TPNH and DPNH.

(Received March 6, 1962; )     

Studies in the Bangioideae III. The Life-history of Porphyra umbilicalis (L.) Kutz. var. lociniata (Lightf.) J. Ag.: A. The Conchocelis-Phase in culture

As the first step in an investigation of the life-history ofPorphyra umbilicalis (L.) Kütz. var. laciniata (Lightf.)J. Ag., spores which have originated as a result of the repeateddivision of the mother-cells have been germinated. When grownon glass their method of germination and growth agrees withthat described by previous investigators, but when germinatedon sterile shell the germ-tubes penetrate the shell and developinto growths identical with Conchocelis rosea Batters. This‘species’, therefore, is a phase in the life-historyof P. umbilicalis var. laciniata and not an autonomous species.The development of the ‘Conchocelis’-phase in cultureis described and also the formation of fertile cell-rows and‘plantlets’. Until the significance of these structuresis understood and a cytological investigation completed, therelationship of the intertidal leafy Porphyra-thallua to thefilamentous, shell-inhabiting, deep-water Conchocelis cannotbe expressed in the terms usually employed to denote the variousphases of the life-histories of algae.     

ROLE OF SULFUR IN THE CELL DIVISION OF CHLORELLA, WITH SPECIAL REFERENCE TO THE SULFUR COMPOUNDS APPEARING DURING THE PROCESS OF CELL DIVISION II.

1. Chlorella cells, which had been grown synchronously undersulfur-deficient conditions and thus rendered unable to performcell division, were made capable of nuclear and cellular divisionby being supplied with ³⁵S-labeled sulfate and nitrate underphotosynthesizing conditions, and the fate of sulfur duringthese recovery processes was followed. 2. When the S-starved cells were provided with sulfate aloneunder photosynthesizing conditions, cells grew appreciably inmass performing nuclear division but remaining incapable ofcellular division. During these processes most of the ³⁵S wasfound to be incorporated into the protein fraction of algalcells. 3. When the cells which had been stalemated at the above-mentionedstage were supplied with nitrate, they grew further in massand eventually performed cellular division. During this periodthe ³⁵S was found to be distributed not only in the proteinfraction, but also in an appreciable amount in the cold andhot acid-soluble fractions. 4. By paper-electrophoretic experiments it was found that thenature of the sulfur substances appearing in the hot acid-solublefraction changed strikingly during the process of cellular division.Zone electrophoresis and an anion-exchange chromatography ofthese substance isolated from the cells at the completion ofcellular division, disclosed that they were most probably deoxypentosepolynucleotides containing sulfur in some form yet unidentified. 5. It was demonstrated that there exist some antagonistic relationsbetween the protein synthesis and the formation of these sulfur-containingdeoxypentose polynucleotides, and that the former predominatesunder photosynthesizing conditions while the latter outweighsunder nonphotosynthesizing conditions. (Received August 9, 1960; )     

Pollen-Derived Haploids of Nicotiana Knightiana, N. raimondii, and N. attenuata

Haploid plantlets were obtained in large numbers in three diploid,24-chromosome species of Nicotiana by culture of anthers ator just past the first pollen mitosis. The three species wereN. Knightiana, N. raimondii, and N. attentiata. Efficiency ofhaploid production varied from about 10 per cent in N. attenuatacultures to 30 and 38 per cent respectively in cultures of N.raimondii and N. Knightiana. H-medium without hormones and standardcultural conditions were used. N. Knightiana appeared to beespecially suitable for haploid studies on account of its highplantlet productivity, low chromosome number, and distinctivekaryotype.     

Growth and development in Phalaris III. Effect of temperature on critical daylength in geographic strains of P. tuberosa L.

The critical daylength for flowering in Phalaris tuberosa L.,a cold-requiring, long-day plant, increases with increasingtemperature. The significance of this phenomenon is discussedin relation to the control of flowering time and the photoperiodicresponse mechanism. (Received November 5, 1968; )     

Water Relations of Tropical Epiphytes: I. RELATIONSHIPS BETWEEN STOMATAL RESISTANCE, RELATIVE WATER CONTENT AND THE COMPONENTS OF WATER POTENTIAL

The water relations of five species of tropical vascular epiphytesnative to Malaysia were studied. The species were ferns: Pyrrosiaadnascens (Forst.) Ching. and Pyrrosia angustata (Sw.) Ching.;orchids: Eria velutina Lindl., Dendrobium tortile Lindl. andDendrobium crumenatum Sw. Leaf resistance as a function of leafwater potential was measured for the two ferns. The criticalwater potential at which stomata closed was found to be highin each case; –0.75 MPa and –0.5 MPa respectively.The components of water potential were estimated with the pressurechamber as functions of relative water content. For each speciescell sap was found to be dilute, pressure potential low at fullturgor, and the change in relative water content between fullturgor and wilting point small. Small values of solute potentialat full turgor were also found for the ferns and E. velutinausing a vapour pressure osmometer. Values of the bulk modulusof elasticity of the leaf tissue for each species lay withinthe range of published data. The significance of these resultsfor the epiphytic way of life is discussed. Key words: Water potential, Epiphytes, Diffusive resistance, Orchid, Fern     

Metabolic Responses to Sulfur in Lucinid Bivalves

SYNOPSIS. Lucinid bivalves occur widely in habitats rangingfrom subtidal to deep ocean basins. The evidence reported todate supports the contention that all lucinids contain intracellular,sulfur-oxidizing bacterial symbionts which contribute substantiallyto the nutrition of the intact association. These burrowingbivalves are found in both high and low sulfide habitats. Examinationsof sulfur compounds in the hemolymph of lucinids reveal thatthiosulfate may be a key intermediate in the metabolism of sulfidein all members of this family. The presence of free sulfidein the hemolymph of both freshly collected and experimentalclams suggests that the total sulfide oxidation/detoxificationcapacity of the lucinids may be substantially lower than thatfound in other sulfur bacteria/invertebrate symbioses. Intracellulargranules catalyze the oxidation of sulfide in L. floridana,one apparent line of sulfide toxicity defense in this species.These electron-dense granules occur in high densities in thebacteriocytes (bacteria-containing gill cells) of all lucinids.Thiosulfate produced during this detoxification may be availablefor further oxidation by the abundant symbionts housed in thesesame cells. Hemolymph thiol and elementalsulfur levels in clamsfrom low sulfide habitats suggest that these animals have evolvedmechanisms for sulfur acquisition. In sulfur-free seawater,sulfur-starved Lucina floridana are infrequent, periodic ventilators,but in the presence of sulfur show a significant increase inventilation frequency. This periodic ventilation may be a traitof all lucinids that plays a substantial role in reducing metaboliccosts during low nutrient availability.     

Salt Tolerance in the Halophyte, Suaeda maritima (L.) Dum.: Properties of Malic Enzyme and PEP carboxylase

Malic enzyme and phosphenol pyruvate carboxylase activitieshave been isolated and characterized from the shoots of Suaedamaritima plants grown in culture solution (with and withoutNaCl) or in tap water. The enzymes isolated from the lattershowed increases in both specific activity and K_m values incomparison with plants grown in culture solution: however, theaddition of NaCl to the culture solution had no significanteffect on either enzyme. Malate levels were high in plants grownin tap water, reduced by an ordeT of magnitude by the additionof culture solution and then halved by the addition of NaCl. Both enzymes were inhibited in vitro by NaCl, although the additionof high concentrations of betaine and proline to the assay mediumdid not further inhibit enzyme activity. The significance ofthese results is discussed in relation to the proposed roleof betaine and proline as cytoplasmic osmoregulators. Suaeda maritima, halophyte, salt tolerance, malic enzyme, PEP carboxylase     

Phosphate Deficiency in Maize. II. Enzyme Activities

Low-phosphate (P) treatment decreased photosynthetic rates ofmaize plants (Zea mays L.) by about 50% 18 to 19 days afterplanting [Usuda and Shimogawara (1991) Plant Cell Physiol. 32:497]. Low-P treatment decreased the enzyme activities differentially(by 0-49%). The significance of the decreased activities ofpyruvate.Pj dikinase (by 29%), phosphoenolpyruvate carboxylase(by 49%), and ribulose 1,5-bisphosphate carboxylase (by 42%)in the detrimental effects of low-P treatments on the ratesof photosynthesis is discussed. (Received August 9, 1991; Accepted October 1, 1991)     

Promotion of Cocklebur Seed Germination by Allyl, Sulfur and Cyanogenic Compounds

The effects of allyl, sulfur and cyanogenic compounds on thegermination of upper cocklebur (Xanthium pennsylvanicum Wallr.)seeds were examined. Mercaptoethanol and methylmercaptan aswell as KCN, substrates for rßcyanoalanine synthase(CAS), and H₂S and thiocyanate, the products of the CAS catalyzingreaction, were effective in promoting germination, suggestingthe involvement of CAS in germination. Most of allyl compounds, especially allylthiourea, as well asethylene which activated CAS [Hasegawa et al. (1994) Physiol.Plant. 91: 141], promoted the germination in an abnormal typewhich occurred by the predominant growth of cotyledons as didC₂H₄ [Katoh and Esashi (1975) Plant Cell Physiol. 16: 687].However, they failed to activate CAS unlike ethylene, and toliberate free ethylene during an incubation period. It was thuspossible that an C₂H₄-like double bond within allyl compoundscan act to promote seed germination. (Received June 10, 1996; Accepted August 21, 1996)