NUTRITIONAL STUDIES OF THE EDIBLE SEAWEED PORPHYRA TENERA I. THE INFLUENCE OF DIFFERENT B12 ANALOGUES, PLANT HORMONES, PURINES AND PYRIMIDINES ON THE GROWTH OF CONCHOCELIS

The red alga Porphyra tenera has been obtained in axenic cultureby the "dip and drag" technique in an agarized medium containingantibiotics. In the axenic culture, the Conchocelis of P. teneraneeds vitamin B₁₂ for growth. The addition of other vitaminsdoes not increase growth further. The pattern of specificityis similar to that of Escherichia coli 113-3. Factor B, pseudovitaminB₁₂ and Factor Z₂ support as much growth as vitamin B₁₂, while2-methylmercaptoadenine and 5-methyl benzimidazole cobalamineincrease the growth more than B₁₂. All the analogues containingbenzimidazole can replace B₁₂. Kinetin, adenine, indoleaceticacid and especially gibberellic acid increase growth. Threepurines, (xanthine, hypoxanthine and guanine) and three pyrimidines(uracil, methylcytosine and thymine) also promote growth inthe presence of vitamine B₁₂. (Received January 18, 1965; )     

Vitamins, phytoplankton and bacteria: symbiosis or scavenging?

The conclusion that over 25% of global primary production dependson direct algal/bacterial symbiosis involving vitamin B₁₂ [Croftet al., (2005) Algae acquire vitamin B₁₂ through a symbioticrelationship with bacteria. Nature, 438, 90–93] is patentlyfalse, for it is based on a misconception of the probable levelof the vitamin B₁₂ requirement in marine pelagic algae. A reviewof the various published attempts at measuring this requirementsuggests that it is likely to be so low that oceanic and coastalconcentrations of the vitamin would usually be sufficient tosustain the populations that occur without the assistance ofdirect algal/bacterial symbiosis. The levels measured are discussedin relation to method (batch or continuous culture) and protocolsused. Requirement values considered by the author to be acceptablerange from 0.1 to 0.3 pM for the vitamin growth saturation constant(K_S) and from 30 to 100 µL algal biomass pmol^–1vitamin for the yield.     

Synthesis of Receptor Sites for Vitamin B12 in Euglena gracilis

In Euglena gracilis, vitamin B₁₂ uptake follows a biphasic patternconsisting of an initial rapid phase followed by a slower secondaryphase. Chase experiments showed that vitamin B₁₂ was tightlybound to its receptor-sites during either rapid or slow phaseof uptake. The slow phase was markedly inhibited when Euglenacells were preincubated with cycloheximide for 30 min at a concentrationof 100µg/ml. When the preincubation time was longer than30 min, a gradual inhibition of the rapid phase occurred andreached 84% after 4 h. This inhibitory effect of cycloheximideis reversible. On the other hand, tunicamycin at 1 µ/mlirreversibly inhibited the B₁₂ uptake suggesting that the receptor-sitefor B₁₂ is glycoprotein in nature. These results suggest thatthe rapid phase is also dependent on protein synthesis and representsB₁₂ binding on preexisting free receptors whereas the secondaryslow phase represents B_l2 binding on newly synthesized receptors.Both phases of uptake seem to be controled by the same receptor.The half-life of the free receptor is estimated to be 66 minwhereas the B₁₂ receptor complex is very stable. (Received October 3, 1988; Accepted February 15, 1989)     

Interspecific growth regulation in species succession through Vitamin B12 competitive inhibition

The growth response of Skeletonema costatum to vitamin B₁₂ wascompetitively inhibited by exudate from either Gonyaulax tamarensisor Cyclotella cryptica at vitamin concentrations and cell densitiespossible in the aquatic environment. Both species produce sufficientquantities of the inhibitor to suggest that it is an importantecological factor affecting species composition. Although theexact chemical structure is not known, it has a high molecularweight as determined by ultra-filtration and can be denaturedby autoclaving in buffered media at pH 7.6.     

A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway

Vitamin B₆ is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B₆ and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B₆ homeostasis. Due to the water-soluble nature of vitamin B₆ and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B₆ in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na⁺ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant (K_m) of 11.99 ± 1.41 µM and a maximal velocity (V_max) of 67.63 ± 3.87 pmol · mg protein^-1 · 3 min^-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant (K_i) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca⁺²/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the V_max, but not the apparent K_m, of the pyridoxine uptake process. These results demonstrate, for the first time, the involvement of a specialized carrier-mediated mechanism for pyridoxine uptake by intestinal epithelial cells. This system is pH dependent and amiloride sensitive and appears to be under the regulation of an intracellular PKA-mediated pathway. vitamin B₆; intestinal transport; transport regulation; Caco-2 cell     

The Cobalt Requirement of Non-legume Root Nodule Plants

As already shown for Alnus glutinosa, cobalt is found to beessential for the proper growth of nodulated plants of Casuarinacunninghamiana and Myrica gale in a nitrogen-free rooting medium.If cobalt is not supplied, the plants develop symptoms of nitrogendeficiency; under the conditions of the experiments such symptomsbecame pronounced during the second season of growth of theseperennial plants. No cobalt requirement could be detected innon-nodulated plants of Alnus and Myrica supplied with nitrateor ammonium-nitrogen, and this suggests that in nodulated plantsthe need for cobalt is confined to the nodules. Vitamin B₁₂analogues are shown to be present in the nodules in relativelylarge amounts when cobalt is supplied, their formation beingattributed to the endophytes, which may therefore require cobaltfor their growth. The great reduction in fixation of atmosphericnitrogen in cobalt-deficient nodules may be due to a retardedgrowth of the endophyte, though this is not the only possibility.The cobalt relation of these non-legumes appears to be basicallysimilar to that of legumes.     

Microbial and enzymatic production of l-carnitine

l-Carnitine (vitamin B_T) plays a vital role in the transportation of long-chain fatty acids into mitochondrial matrix in mammals. l-Carnitine has a wide range of applications in pharmaceuticals, food products, and feed additives. Due to the increasing demand for l-carnitine in food and pharmaceutical applications, production of this compound has become prominent. However, very little has been reported on the production of l-carnitine. This review discusses the microbial and the enzymatic production of l-carnitine from different starting materials (substrates).     

Changes in levels of Proteases and Their Inhibitors during Development of the Seeds of Job's Tears (Coix lacryma-jobi L. var. Ma-yuen Stapf) and Their Localization and Interactions

Changes in levels and localization of proteases and a trypsininhibitor (JBTI) in the developing seeds of Job's tears (Coixlacryma-jobi L. var. Ma-yuen Stapf) were followed, and theirinteractions were examined. The JBTI was induced from the middlestage of development (12 DAF, i.e., 12 days after flowering)and increased until the late stage of development (24 DAF),and was localized in the germ. Two groups of proteases (A, thosewith molecular weights of 55–70 kDa; and B, those withmolecular weights greater than 94 kDa) were detected by activestaining of substrate-containing SDS-polyacrylamide gels. Thegroup of larger proteases seems to consist of three members(B₁, B₂₎ B₃, with increasing molecular weights). Band A wasobserved only in the early stage of development (until DAF 9),band B₂ persisted during all stages, while bands B, and B₃ werepresent only during early and late stages, respectively. Thegroup A proteases and one of the group B proteases (probablyB₁) were inhibited by diisopropyl-fluorophosphate (DFP), bytrypsin inhibitors from soybean and rice, and by JBTI. The proteasesthat were present in the seeds of Job's tears at a late stageseemed to be localized in the germ. (Received September 9, 1988; Accepted April 19, 1989)     

The distribution of cobalt inT. subterraneum

Summary The distribution of cobalt in plants ofT. subterraneum grown on different levels of supply of cobalt and nitrate nitrogen has been measured, and the nodules and to a lesser extent the roots have been found to accumulate much more than the leaves.The distribution of vitamin-B₁₂-type compounds and of Co⁶⁰ in the nodule have also been determined following fractionation of the nodule components by hgh speed centrifugation. Most of the cobalt whether present as vitamin B₁₂, or as indicated by the measurement of Co⁶⁰, is present in the supernatant and in the bacteroids. The distribution of Co⁶⁰ is much the same in the nodules from both effective and non-effective strains. The quantities of vitamin B₁₂ present in both bacteroids and the supernatant are greatly increased, in relation to the fresh weight of the nodule tissue, when the supply of cobalt in the nutrient is raised.When Co⁶⁰ is incorporated into the plant a proportion of the activity is found to be present as vitamin B₁₂, free from bacteroids, within four days of addition. After this time the supernatant still contained about 90 per cent of the activity that entered. Of this some 40 per cent was present as ionic cobalt, 19 per cent as vitamin B₁₂ like compounds, and the remainder as a compound, presently termed Factor N, which is so far unidentified.Most of the data on which this paper is based are from the thesis submitted by S.B.W. for the degree of Ph. D. of the University of Nottingham.     

Scaling of submaximal oxygen uptake with body mass and combined mass during uphill treadmill bicycling

This study examined the scaling relationships ofnet O₂ uptake [O_2(net) = O₂  restingO₂] to body mass(M_B) andcombined mass (M_C = M_B + bicycle)during uphill treadmill bicycling. It was hypothesized thatO_2(net)(l/min) would scale proportionally withM_C [i.e.,O_2(net)  M^1.0_C] and less than proportionally withM_B [i.e.,O_2(net)  M_B].Twenty-five competitive cyclists [73.9 ± 8.8 and 85.0 ± 9.0 (SD) kg forM_B andM_C,respectively] rode their bicycles on a treadmill at 3.46 m/s andgrades of 1.7, 3.5, 5.2, and 7.0% whileO₂ was measured. Multiplelog-linear regression procedures were applied to the pooledO_2(net)data to determine the exponents forM_C andM_B afterstatistically controlling for differences in treadmill grade anddynamic friction. The regression models were highly significant (R² = 0.95, P < 0.001). Exponents forM_C (0.99, 95%confidence interval = 0.80-1.18) andM_B (0.89, 95%confidence interval = 0.72-1.07) did not differ significantly fromeach other or 1.0. It was concluded that the 0.99 M_C exponent wasdue to gravitational resistance, whereas theM_B exponent was<1.0 because the bicycles were relatively lighter for heaviercyclists.

     

Oxidative Damages and Repair in Euglena gracilis Exposed to Ozone II. Membrane Permeability and Uptake of Metabolites

Significant injuries to the plasma membrane were detected inEuglena gracilis cells during ozone exposure (240 µ1.liter¹,delivery rate of l µmol.min^–1), as assessed by measuringthe alterations of vitamin B₁₂ and acetate uptakes and the leakageof intracellular K⁺ (Rb⁺). A rapid decrease in the uptake ofvitamin B₁₂ and acetate was observed within 15 min of treatment,indicating that both transport systems are very sensitive toO₃. On the other hand, the leakage of intracellular K⁺ ions,as measured by the efflux of ⁸⁶Rb⁺ from prelabelled cells, couldonly be detected after 30 min of O₃ exposure. These resultssuggest that the initial metabolic symptoms of injury is atthe level of the two transport systems examined and that thealteration of the membrane permeability to K⁺ ions appears asa second step in the cascade of oxidative events at the plasmamembrane level. When Euglena cells were allowed to recover underautotrophic growth conditions following O₃ treatment, vitaminB₁₂ and ⁸⁶Rb⁺ (K⁺) ions uptakes returned gradually to controllevel within 5 h of the recovery period. Acetate uptake returnedto control level at a slower rate and needed 20 h for completerecovery. These results indicate that the cells were able toactively repair most of the initial oxidative damages inducedby O₃. The metabolic significance of the repair mechanism(s)is discussed. (Received December 25, 1989; Accepted July 23, 1990)     

Variation in the DNA Content of Glycine Species

Nuclei were isolated from cotyledons of a range of accessionsfrom 14 species of Glycine. These were stained with ethidiumbromide and the relative fluorescence for each genotype wasmeasured by flow cytometry. The DNA content was estimated bycomparison of relative fluorescence with that from nuclei fromseedling leaves of Allium cepa, whose DNA content has been calculatedpreviously by chemical assay. The 4C amounts for diploid Glycineranged from 3.80 to 6.59 pg. Two groups of diploid species appearedfrom the analysis. The first consisted of species with amountsranging from 3.80 to 5.16 pg and included G. canescens (AA),G. argyrea (A₁ A₁), G. clandestina (A²A²), G. microphylla(BB),G. latifolia (B₁B₁), G. tabacina 2n=40 (B²B²), G. tomentella2n=38 (EE) and 2n=40 (DD), G. max and G. soja (GG), G. arenariaand G. latrobeana. A second group had higher DNA contents rangingfrom 5.27 to 6.59 pg, and consisted of G. curvata, G. cyrtoloba(CC), and G. falcata (FF). The polyploid species, G. tabacina2n=80 (AABB, BBB¹B¹), G. tomentella 2n=78 and 2n=80 (AAEE andDDEE, respectively) contained amounts approximating to the sumsof the respective parental diploid species thought to have givenrise to these allotetraploids. Intraspecific variation was detectedin the DNA content of G. canescens. Within the overall distributionof DNA amounts found in A genome species, each genome containeda range of DNA contents specific to that species. This phenomenonwas also detected amongst B genome species.     

Effects of Chemicals Often Regarded as Germination Stimulants on Seed Conditioning and Germination of Witchweed (Striga asiatica)

Gibberellin A₃ (GA₃), kinetin and zeatin promoted germinationof conditioned witchweed (Striga asiatica [L.] Kuntze) seedssimilar to dl-strigol and a natural stimulant from corn rootexudates. GA₃ promoted germination only with scarified seeds.However, these regulators, as well as dl-strigol and naturalstimulants, inhibited the conditioning of seeds and their subsequentgermination. On the other hand, abscisic acid induced 74% germinationof conditioned seeds but had no effect on seed conditioning.Other chemicals such as sucrose, NaNO₃, vitamin B₁, L-methionine,nicotinamide, indoleacetic acid and colchicine had no apparenteffect on seed conditioning, while sucrose, L-methionine andinositol induced about 30 % germination of conditioned seeds.Seeds generally required either a longer conditioning time ora higher terminal dl-strigol concentration to eliminate anyinhibition of seed conditioning induced by a compound. dl-Strigol, germination stimulation, parasitic plants, seed conditioning, seed germination, Striga asiatica (L.) Kuntze, weed control, growth regulating substances     

Mechanisms of Electron Flow through the QB Site in Photosystem II. 3. Effects of the Presence of Membrane Structure on the Redox Reactions at the QB Site

Reduction kinetics of chloro- or methyl-substituted benzoquinones(BQs) in spinach PSII membrane fragments or n-heptyl-rß-D-thioglucoside-extractedPSII core complexes (HTG-PSII) was studied and compared to thatin cyanobacterial PSII core complexes [Satoh et al. (1995) PlantCell Physiol. 36: 597]. It was found that the BQs accept electronsat two sites (the Q_B and PQ sites) in the both spinach preparationsas in the cyanobacterial preparation. Maximum turnover rates(V_max) and binding affinities (K_m) of the two sites were estimated.Comparison of the values in PSII membrane fragments with thosein HTG-PSII showed that removal of the membrane structure orlight-harvesting chlorophyll a/b protein complexes from thePSII core complexes had little effect on the characteristicsof the Q_B site, indicating that the HTG-PSII have the intactQ_B site and are good materials to study the site. The K_m andV_max values were comparable to those in the cyanobacterial preparation. Low affinity (high K_m values) and high V_max values of methyl-substitutedBQs to the Q_B site and almost the same rate of intrinsic electronflow through the Q_B site in the both spinach preparations furthersupport the hypothesis that the plastoquinone (PQ) moleculeat the Q_B site is not replaced by another PQ molecule but, afterreduction, only its head group goes out of the Q_B pocket, andcomes back after transferring the electrons and protons to afree PQ molecule [Satoh et al. (1993) Z. Naturforsch. 48c: 174]. (Received May 2, 1996; Accepted July 26, 1996)     

Photosynthetic parameters, pigment composition and respiration rates of the marine diatom Skeletonema costatum grown in continuous light and a 12:12 h light-dark cycle

Nutrient-sufficient cultures of a Trondheimsfjord (Norway) cloneof the marine centric diatom Skeleionema costatum (Grev.) Clevewere grown at 75 µmol m^–2 s^–1 and 15C at24 and 12 h daylength to study diurnal variations and the effectof daylength on pigment and chemical composition, photosyntheticparameters, dark respiration rates and scaled fluorescence excitationspectra (F), the latter used as estimates for the absorptionof energy available to Photosystem II. Specific growth rateswere 1.06 and 0.56 day^– in 24 and 12 h daylength, respectively,while dark respiration rates were generally 85% of the net growthrate. The Chla-normalized photosynthetic coefficients P^B_m anda^B were {small tilde}20–25% higher in continuous lightthan at 12 h daylength, while the Chla:C ratio was {small tilde}15%lower (0.051 versus 0.061 w:w). Thus, the carbon-normalizedcoefficients P^c_m and a^c were <11% lower at 24 h than at 12h daylength. The maximum quantum yield _max, the Chla:C ratioand F differed negligibly, as did the light saturation indexl_k, the N:C ratio and the ratios Chlc:Chla and Fucoxanthin:Chla. P^B_m and l_k did not exhibit diurnal variations at 24 hdaylength, and varied within 23% of the daily mean at 12 h daylength.Predictions of the daily gross photosynthetic rate based ondata for a given time of the day should thus not be >10%in error relative to an integrated value based on several datasets collected through 24 h. _max was 0.084–0.117 mol O₂(mol photons)^– for gross oxygen evolution. However, ifused in mathematical models for predicting the gross and netgrowth rates (i.e. the gross and net carbon turnover rates),‘practical’ values of 0.076 and 0.040 g-at C (molphotons)^–, respectively, should be employed. Correspondingly,values for a^B and P^B_m should be adjusted pro rata. ¹Present address: College of Marine Studies, Sjmannsveien 27,N-6008 lesund, Norway     

Nutritional and Other Aspects of Fruit-body Production in Pure Cultures of Collybia velutipes (Curt.) Fr.

Fruit-body production of Collybia velutipes on a synthetic mediumconsisting of minerals, asparagine, sucrose, and vitamin B₁has been studied. Compared with others tested this medium favouredfructification rather than mycelium growth. Primordia aroseon culturea between pH 5•2 and 7•2 approximately.Relatively low initial concentration of asparagine, high initialconcentration of sucrose, and increases in volume (without essentialincrease of the air interface) of media resulted in enhanceddry weights of sporophore crops. These factors variously affectedmycelium yields and the times of primordium production. Detailsof growth, sporophore production, maximum yield, and changesin the medium are given.     

Subunit Proteins of Photosystem I

Photosystem I (PS I) is a supramolecular complex in thylakoidmembranes and mediates the light-driven electron flow from plastocyanin(or cytochrome c₅₅₃) to ferredoxin. It has been establishedthat the PS I complex consists of more than 10 different subunitproteins that ligate 100 to 200 molecules of chlorophylls includingP700, two molecules of phylloquinone and three iron-sulfur centers(F_X, F_A, F_B). The identity and properties of these PS I subunitproteins have been extensively studied, and their genes haverecently been cloned and mutagenized. The current status ofthese investigations is summarized. (Received May 8, 1992; )     

Pulmonary clearance of circulating endothelin-1 in dogs in vivo: exclusive role of ETB receptors

Dupuis, Jocelyn, Carl A. Goresky, and Alain Fournier.Pulmonary clearance of circulating endothelin-1 in dogs in vivo: exclusive role of ET_B receptors.J. Appl. Physiol. 81(4):1510-1515, 1996.The pulmonary circulation plays an importantrole in the removal of circulating endothelin-1 (ET-1). Plasma ET-1levels are increased in pulmonary hypertensive states of variousetiologies (e.g., idiopathic, heart failure, and congenital anomalies)in proportion to the severity of pulmonary hypertension. It is possible that reduced pulmonary clearance of this peptide contributes to thehyperendothelinemia of those pathologies. TheET_A andET_B receptors are abundant in lungtissues: on the vascular endothelium, theET_B receptor is predominant andmay contribute to ET-1 extraction through receptor-mediatedendocytosis. We designed experiments to determine and quantify theimportance of the ET_A andET_B receptors in the pulmonaryextraction of circulating ET-1 in anesthetized dogs. The single-passcumulative tracer ET-1 extraction by the lung was measured with theindicator-dilution technique before and 5 min after intrapulmonaryinjection of the specific ET_Aantagonist BQ-123 (n = 5, 120-960nmol) and the specific ET_Bantagonist BQ-788 (n = 6, 1,000 nmol).The inhibitors had no significant effect on pulmonary and systemichemodynamics. Mean cumulative pulmonary ET-1 extraction was notmodified by BQ-123 [control (C): 36 ± 4%, antagonist (A): 34 ± 6%] but was completely abolished by BQ-788 (C: 34 ± 6%, A: 0 ± 2%, P < 0.001). Thepulmonary rate constant (K) for ET-1removal was also unaffected by BQ-123 (C: 0.050 ± 0.0085 s¹, A: 0.047 ± 0.012 s¹) but significantlydecreased and became close to zero after BQ-788 (C: 0.058 ± 0.014 s¹, A: 0.009 ± 0.007 s¹,P < 0.001). We conclude that theET_B receptor is completely andexclusively responsible for pulmonary ET-1 removal in vivo. Futurestudies are needed to show whether desensitization or downregulation ofthe ET_B receptor may contribute tothe increase in circulating ET-1 levels in conditions associated withpulmonary hypertension. This novel pulmonary endothelial cell functionmay play a protective role by modulating circulating ET-1 levels in thesystemic circulation.

     

Hypoxia- and normoxia-induced reversibility of autonomic control in Andean guinea pig heart

León-Velarde, Fabiola, Jean-Paul Richalet, Juan-CarlosChavez, Rachid Kacimi, Maria Rivera-Chira, José-Antonio Palacios, and Daniel Clark. Hypoxia- and normoxia-induced reversibility ofautonomic control in Andean guinea pig heart. J. Appl.Physiol. 81(5): 2229-2234, 1996.We hereindescribe the regulation of cardiac receptors in a typical high-altitudenative animal. Heart rate response to isoproterenol(HR_Iso)(beats ^· min^{1 ·} mgIso ^· kg¹)and atropine, the density of -adrenergic(_AR) and muscarinic (M₂) receptors, and theventricular content of norepinephrine (NE) and dopamine (DA) werestudied in guinea pigs (Caviaporcellus). Animals native to Lima, Peru (150 m) werestudied at sea level (SL) and after 5 wk at 4,300-m altitude (SL-HA).Animals native to Rancas [Pasco, Peru (4,300 m)] werestudied at high altitude (HA) and after 5 wk at SL (HA-SL). HA animalshad a lower HR_Iso, maximum numberof _AR binding sites(B_max),_AR dissociation constant (K_d), NE, andDA (P < 0.05) and a higherM₂B_max(P < 0.001) when compared with theSL group. HA-SL showed an increase of theHR_Iso, _ARK_d, and NE(P < 0.05) and a decrease of theM₂B_max andK_d (P < 0.0001) when compared with theHA group. The present study demonstrates the differential regulationand reversibility of the autonomic control in the guinea pig heart.

     

Vitamin B12-dependent methionine synthesis in Rhizobium meliloti

Methionine, among the various additions to the medium, could only replace cobalt ion or vitamin B₁₂ required for the growth of $\text{Rhizobium meliloti}$. It was demonstrated that there exists a vitamin B₁₂-dependent terminal step in the methionine synthesis, that is, N⁵CH₃-tetrahydrofolate-homocysteine transmethylase, which can also catalyze the methyl transfer from CH₃B₁₂ to homocysteine, in the cell-free extracts of Rhizobium meliloti. These facts seem to indicate that the vitamin B₁₂-dependent pathway to methionine functions mainly among the B₁₂-dependent enzymatic systems in the wild-type symbionts and this is the chief nutritional significance of cobalt.