Seasonal and Spatial Distribution of Vitamin B12 in a Coastal Area of Eastern Mediterranean Sea

The seasonal and spatial distribution of vitamin B₁₂ was investigated in Eastern Mediterranean (Saronicos Gulf, Aegean Sea). Vitamin B₁₂ concentration had an annual range 0.12–7.93 ng. 1⁻¹ and an annual mean 1.88 ng. 1⁻¹. The statistical analysis of data indicated significant seasonal and regional variations of vitamin B₁₂ in this area and an inverse relationship of this vitamin with chl a. The results showed that the Eastern Mediterranean Sea contains less vitamin B₁₂ than the Western Mediterranean Sea.     

Production of 3‐hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ΔdhaTΔyqhD which can produce vitamin B12 naturally

3‐Hydroxypropionic acid (3‐HP) is an important platform chemical that can be used to synthesize a range of chemical compounds. A previous study demonstrated that recombinant Escherichia coli stains can produce 3‐HP from glycerol in the presence of vitamin B₁₂ (coenzyme B₁₂), when overexpressed with a coenzyme B₁₂‐dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3‐HP in recombinant Klebsiella pneumoniae strains, which naturally synthesizes vitamin B₁₂ and does not require supplementation of the expensive vitamin. The NAD⁺‐dependent gamma‐glutamyl‐gamma‐aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B₁₂ addition, the recombinant K. pneumoniae ΔdhaTΔyqhD overexpressing PuuC could produce ～3.8 g/L 3‐HP in 12 h of flask culture. However, this was possible only under the appropriate aeration conditions; 1,3‐propanediol (1,3‐PDO) (instead of 3‐HP) was mainly produced when aeration was insufficient, whereas a very small amount of both 3‐HP and 1,3‐PDO were produced when aeration was too high. The production of a small amount of 3‐HP under improper aeration conditions was attributed to either slow NAD⁺ regeneration (under low aeration) or reduced vitamin B₁₂ synthesis (under high aeration). In a glycerol fed‐batch bioreactor experiment under a constant DO of 5%, the strain, K. pneumoniae ΔdhaTΔyqhD, overexpressing both PuuC and DhaB could produce >28 g/L 3‐HP in 48 h with a yield of >40% on glycerol. Only small amount of 3‐HP was produced when cultivation was carried out at a constant aeration of 1 vvm or constant 10% DO. These results show that K. pneumoniae is potentially useful for the production of 3‐HP in an economical culture medium that does not require vitamin B₁₂. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of 3‐HP while using this strain. Biotechnol. Bioeng. 2013; 110: 511–524. © 2012 Wiley Periodicals, Inc.     

UNEXPECTED RESPONSE TO VITAMIN B12 OF DOMINANT CENTRIC DIATOMS FROM THE SPRING BLOOM IN THE GULF OF MAINE (NORTHEAST ATLANTIC OCEAN)1,2

Twelve clones (seven species) representative of centric diatoms dominant in the spring phytoplankton bloom in the Gulf of Maine were isolated and rendered axenic. Genera included were Thalassiosira, Porosira and Chaetoceros. Unlike most centric diatoms studied previously, none of these has an absolute requirement for vitamin B_12. However, B₁₂ (5 ng.l^-1) stimulated growth of most clones by eliminating or reducing the lag phase and increasing the growth rate. Bloom population densities developed 4–54 days earlier with B₁₂ present. Several clones grown with B₁₂ removed more than 80% of the vitamin from the medium. When grown in vitamin-free medium the cells put 0.01–0.7 ng.l^-1 B₁₂ into the medium. We conclude that vitamin B₁₂ is of ecological significance even though the requirement for it is not absolute.     

A SURVEY OF AUXOTROPHY IN FIVE FRESHWATER DINOFLAGELLATES (PYRRHOPHYTA)1

Peridiniopsis polonicum (Wolosz.) Bourrelly requires vitamin B₁₂, and Peridinium limbatum (Stokes) Lemm. requires thiamin for growth. Unlike marine Peridinium species, Peridinium willei Huit.-Kaas, P. volzii Lemm., and P. inconspicuum Lemm. do not display auxotrophy. Peridinium volzii is strongly inhibited by concentrations of biotin above 1 μg L^?1.     

Modification of Protein Synthesis by Vitamin B12 in the Marine Algal Flagellate Neochloris pseudoalveolaris*

SYNOPSIS. A strain of the marine algal flagellate Neochloris pseudoalgeolaris required only an inorganic medium for growth; however, in media containing vitamin B₁₂ it became greener, protein synthesis was modified, and the levels of DNA and RNA increased. By isotope tracer technics, transmethylation of the methyl group of methionine was found to play a role in modulation of protein synthesis governed by the vitamin. These results indicate that vitamin B₁₂ stimulates synthesis of chloroplasts in this algal strain.     

Polymorphism of vitamin B12 [57Col binding proteins in rabbit serum

When rabbit serum labelled with vitamin B₁₂[⁵⁷Co] was subjected to starch gel electrophoresis and au;oradiography, three phenotypes of proteins capable of binding vitamin B₁₂ were observed. Family data revealed that these phenotypes (called TC-A, TC-AB and TC-B) are controlied by two codominant alleles (TC^A and TC^B), at an autosomic locus. Proteins capable of binding vitamin B₁₂ both in vivo and in vitro are commonly referred to as Transcobalamins and can be found in the serum of numerous animal species (for a review, see Glass, 1974; Allen, 1975; Stenman, 1975). Furthermore, Daiger et al. (1975a) have described seven different patterns of vitamin B₁₂ binding proteins which occur in human plasma and which are presumably controlled by four alleles. The present paper describes experiments in which both starch gel electrophoresis and autoradiography are used to identify three phenotypes of rabbit serum proteins responsible for binding vitamin B₁₂ in vitro. It was found that these three phenotypes are controlled by two allelic codominant genes, at an autosomic locus. Individual serum samples (30 μl), obtained from 385 White New Zealand rabbits varying in age from one month to three years, were incubated with 0.1 ng of vitamin B₁₂[⁵⁷Co] (specific activity: 180 μCi/μg; Lot 247; Radiochemical Centre, Amersham, England) at 37°C for 30 minutes. Starch gel electrophoresis and autoradiography were performed as described by Geldermann (1970) and Daiger et al. (1975b), respectively. Electrofocusing (pH range 3.5–9.5) was conducted in the 2117 Multiphor apparatus (LKB, Bromma, Sweden) according to the manufacturer's instructions. The resulting pH gradient was measured with a surface pH electrode (Ingold, Zürich, Switzerland).     

Vitamin B12, a chlorophyll-related analog to pheophytin a from marine brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells

We previously isolated an analog to chlorophyll-related compounds, pheophytin a, from the marine brown alga Sargassum fulvellum and demonstrated that it is a neurodifferentiation compound. In the current study, we investigated the effects of the pheophytin a analog vitamin B₁₂ on PC12 cell differentiation. In the presence of a low level of nerve growth factor (10 ng ml⁻¹), vitamin B₁₂ demonstrated neurite outgrowth-promoting activity in PC12 cells. The effect was dose-dependent in the range of 6–100 μM. In the absence of nerve growth factor, vitamin B₁₂ did not promote differentiation. To investigate the mechanism for this effect, we conducted differentiation assays and western blot analysis with signal transduction inhibitors and found that vitamin B₁₂ did not promote PC12 cell differentiation in the presence of K252a or U0126 inhibitors. These results suggest that vitamin B₁₂ stimulates PC12 cell differentiation through enhancement of the mitogen-activated protein kinase signal transduction pathway, which is also induced by nerve growth factor. Thus, vitamin B₁₂ may be a good candidate for treatment of neurodegenerative diseases such as Alzheimer’s disease.     

GROWTH OF VITAMIN B12-REQUIRING MARINE DIATOMS IN MIXED LABORATORY CULTURES WITH VITAMIN B12-PRODUCING MARINE BACTERIA12

The vitamin B₁₂ requirement of several marine diatoms can be satisfied in B₁₂^?limited laboratory cultures by heterotrophic marine bacteria isolated from the same waters and from sediments. The bacteria can utilize diatom excretory products, or the remains of dead diatom cells, in the production of the vitamin. The growth of 12 B₁₂¹? requiring diatoms (7 genera) in mixed cultures with 14 different bacteria (without added B₁₂) was compared to the growth of those same diatoms in axenic cultures with excess added B₁₂. Diatom growth was generally rapid in the first few days, followed by sustained, slower growth. The diatom yields in mixed cultures ranged from 0.8 to 84% of the yields in axenic cultures with added B₁₂. In a detailed study of one mixed culture, increases in diatom densities were paralleled by increases in cell densities of the bacterium during the first few days of exponential diatom growth. During the period of slow diatom growth, when diatom densities oscillated but steadily increased, the decreases in diatom densities were associated with increased bacterial growth. This suggests that death of a fraction of the B₁₂-limited diatom population releases sufficient organic matter to stimulate growth of the bacteria and their subsequent excretion of B₁₂; this B₁₂ in turn stimulates further growth of the diatoms. Diatom-bacteria interactions leading to the production of B₁₂ may be important in maintaining viable populations of B₁₂-requiring diatoms in nutrient-poor waters during periods between blooms, when conditions are unfavorable for rapid growth.     

Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

Cobalamin (vitamin B₁₂) is a cofactor for essential metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonize the algal phycosphere, forming stable communities that gain preferential access to photosynthate and in return provide compounds such as B₁₂. Extended coexistence can then drive gene loss, leading to greater algal–bacterial interdependence. In this study, we investigate how a recently evolved B₁₂-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B₁₂ producers, its growth in co-culture was slower than the B₁₂-independent wild-type, suggesting that high bacterial B₁₂ provision may be necessary to favour B₁₂ auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B₁₂ makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B₁₂ release provided an advantage in co-cultures. We hypothesize that, given the right conditions, bacteria that release more B₁₂ may be selected for, particularly if they form close interactions with B₁₂-dependent algae.     

Mutualistic interactions between vitamin B(12) -dependent algae and heterotrophic bacteria exhibit regulation

Many algae are auxotrophs for vitamin B₁₂ (cobalamin), which they need as a cofactor for B₁₂‐dependent methionine synthase (METH). Because only prokaryotes can synthesize the cobalamin, they must be the ultimate source of the vitamin. In the laboratory, a direct interaction between algae and heterotrophic bacteria has been shown, with bacteria supplying cobalamin in exchange for fixed carbon. Here we establish a system to study this interaction at the molecular level. In a culture of a B₁₂‐dependent green alga Chlamydomonas nivalis, we found a contaminating bacterium, identified by 16S rRNA analysis as Mesorhizobium sp. Using the sequenced strain of M. loti (MAFF303099), we found that it was able to support the growth of B₁₂‐dependent Lobomonas rostrata, another green alga, in return for fixed carbon. The two organisms form a stable equilibrium in terms of population numbers, which is maintained over many generations in semi‐continuous culture, indicating a degree of regulation. However, addition of either vitamin B₁₂ or a carbon source for the bacteria perturbs the equilibrium, demonstrating that the symbiosis is mutualistic and facultative. Chlamydomonas reinhardtii does not require B₁₂ for growth because it encodes a B₁₂‐independent methionine synthase, METE, the gene for which is suppressed by addition of exogenous B₁₂. Co‐culturing C. reinhardtii with M. loti also results in reduction of METE expression, demonstrating that the bacterium can deliver the vitamin to this B₁₂‐independent alga. We discuss the implications of this for the widespread distribution of cobalamin auxotrophy in the algal kingdom.     

Nutritional effect of freshwater Chlorella on growth of the rotifer Brachionus plicatilis

Mass production of Brachionus plicatilis is usually accomplished by feeding so-called marine Chlorella (Nannochloropsis oculata) to the rotifers in marine fish hatcheries. If the marine Chlorella are in short supply, baker's yeast is usually used as a supplementary food. Recently, a condensed suspension of freshwater Chlorella (Chlorella vulgaris, k-22) was commercially developed as another supplementary food. We have evaluated the dietary value of this freshwater Chlorella for growth of the rotifer by means of individual and batch cultures. Rotifers cultured with the freshwater Chlorella suspension under almost bacteria-free conditions, showed very suppressed growth. However if the Chlorella was supplemented with vitamin B₁₂ by adding the vitamin solution into the suspension or by culturing the Chlorella in a medium containing vitamin B₁₂, the nutritional value of freshwater Chlorella was greatly improved and almost at the same level as that of marine Chlorella. Condensed Chlorella may therefore be effective as a supplementary food if vitamin B₁₂ is supplied.     

Low holo-transcobalamin levels are prevalent in vegetarians and is associated with coronary artery disease in Indian population

Coronary artery disease (CAD) has been increasing alarmingly in India. We had earlier shown that vitamin B₁₂ deficiency is associated with CAD in Indian population. However, only about a quarter of the total vitamin B₁₂ is internalised in the cells by the proteins transcobalamin II. Vitamin B₁₂-bound transcobalamin II (holotranscobalamin, holoTC) is thus referred to as biologically active B₁₂. In this study, we ascertained the levels of holoTC in 501 CAD cases and 1253 healthy controls and for the first time show that holoTC levels are significantly lower (p?=?2.57E-4) in CAD (26.81?pmol/l) cases as compared to controls (29.97?pmol/l).     

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.     

Vitamin B(12) incorporated with multiwalled carbon nanotube composite film for the determination of hydrazine

Electrochemically active composite film containing multiwalled carbon nanotubes (MWCNTs) and vitamin B₁₂ was synthesized on glassy carbon, gold, and indium tin oxide electrodes by the potentiodynamic method. The presence of MWCNTs in the composite film (MWCNT–B₁₂) modified electrode mediates vitamin B₁₂’s redox reaction, whereas vitamin B₁₂’s redox reaction does not occur at bare electrode. The electrochemical impedance spectroscopy studies reveal that MWCNTs present in MWCNT–B₁₂ film enhance electron shuttling between the reactant and electrode surface. The surface morphology of bare electrode, MWCNT film. and MWCNT–B₁₂ composite film was studied using atomic force microscopy, which reveals vitamin B₁₂ incorporated with MWCNTs. The MWCNT–B₁₂ composite film exhibits promising enhanced electrocatalysis toward hydrazine. The electrocatalysis response of hydrazine at MWCNT film and MWCNT–B₁₂ composite film was measured using cyclic voltammetry and amperometric current–time (i–t) curve techniques. The linear concentration range of hydrazine obtained at MWCNT–B₁₂ composite film using the i–t curve technique is 2.0 μM–1.95 mM. Similarly, the sensitivity of MWCNT–B₁₂ composite film for hydrazine determination using the i–t curve technique is 1.32 mA mM⁻¹ cm⁻², and the hydrazine’s limit of detection at MWCNT–B₁₂ composite film is 0.7 μM.     

Studies on the vitamin B12 auxotrophy of Rhodocyclus purpureus and two other vitamin B12-requiring purple nonsulfur bacteria

The vitamin B₁₂ requirement of Rhodocyclus purpureus 6770, Rhodospirillum tenue 1/67, and Rhodopseudomonas palustris G 53/2 was determined. A wide variety of biogenetic precursors of the vitamin including cobinamide, cobyric acid, cobinic acid and several partially amidated cobyrinic acids showed growth-promoting activity in all three strains. In R. purpureus vitamin B₁₂ could even be substituted by cobyrinic acid which is the first cobalt-containing precursor of vitamin B₁₂ so far established. Neither methionine, deoxynucleosides, dimethylbenzimidazole nor increased amounts of cobalt could replace vitamin B₁₂ as growth factor.Cupribalamin, which is a strong antimetabolite of vitamin B₁₂ in Escherichia coli 113-3 and Lactobacillus leichmannii ATCC 7830, exhibited only a weak antagonistic effect on growth of R. purpureus and R. tenue. Growth of R. palustris was not inhibited by cupribalamin. The cells of all three strains were shown to contain metal-free corrinoids in addition to cobalt-containing corrinoids. The principal products were identified as 5-deoxyadenosylcobalamin and hydrogenobalamin, the metal free analogue of vitamin B₁₂. The latter does not originate from the vitamin by removal of cobalt but is de novo biosynthesized as could be demonstrated in the case of R. purpureus by a labelling experiment with [¹³C] methyl-l-methionine.     

VITAMIN PRODUCTION AND UTILIZATION BY PHYTOPLANKTON IN MIXED CULTURE1

The production and utilization of vitamins by 2 or more, marine phytoplankters cultured in the same vessel were demonstrated. The release of toxic materials or vitamin inactivators wax also observed. The utilization of vitamins by those phytoplankters requiring them and the production of toxic materials were determined from increases and decreases in cell numbers of certain algae grown in mixed cultures. Vitamin utilization was most readily observed in mixed cultures where 2 phytoplankters were present. Dunaliella tertiolecta and Skeletonema costatum produced utilizable thiamine for Coccolithus huxleyi. C. huxleyi released utilizable vitamin B₁₂ for Cyclotella nana. D. tertiolecta, Phaeodactylum tricornutum, and S. costatum produced utilizable biotin for Amphidinium carterae. The amount of utilizable vitamin and rate at which it was released depended on the phytoplankters present and conditions of incubation. In complex systems with more than 2 phytoplankters, beneficial effects to utilizers were often noted for short durations during the incubation period. At the end of the experiments the beneficial effects were usually not evident. P. tricornutum (nonvitamin requirer) was stimulated by a mixture of carryover B₁₂ and thiamine when growing with A. carterae, indicating that in vitamin-free media it cannot synthesize vitamin(s) fast enough to allow for a maximum growth rate. The concentrations of vitamins in seawater samples may not be the amounts available to vitamin-requiring organisms. Among the factors affecting vitamin availability are the production of inhibitors and vitamin inactivators by various organisms in the ecosystem.     

PRODUCTION OF VITAMIN B12, THIAMINE,AND BIOTIN BY PHYTOPLANKTON1

Some ecologically important phytoplankters released vitamins into culture medium during growth. Skeletonema costatum and Stephanopyxis turris (vitamin B₁₂-requirers) produced both thiamine (vitamin B₁) and biotin when growing with either 12 or 2 ng vitamin B₁₂/liter. Gonyaulax polyedra (vitamin B₁₂-requirer) produced thiamine with 12 ng vitamin B₁₂/liter, and Coccolithus huxleyi (thiamine-requirer) produced vitamin B₁₂ and biotin with 120 ng thiamine/liter, but only biotin with 10 ng thiamine/liter. The amount of vitamin produced by an alga and rate at which it was produced varied with the phytoplankter, the concentration of the required vitamin, and incubation time. Vitamins produced during early and exponential growth were due to excretions, and those produced at stationary growth resulted from excretion and release due to cell lysis. Uptake of the required vitamin by all phytoplankters was greatest during the first few days of incubation. On continued incubation the rate of uptake/cell decreased. In the sea phytoplankters may contribute a major portion of the amount of dissolved vitamins.     

Competitive immunoassay for analysis of vitamin B(12)

In the current work, direct competitive enzyme-linked immunosorbent assay (ELISA) was developed for derivatized vitamin B₁₂ by generating chicken egg yolk immunoglobulins (IgY) against derivatized vitamin B₁₂ and purified using affinity chromatography. Checkerboard assay was performed with vitamin B₁₂ antibody and vitamin B₁₂–alkaline phosphatase conjugate followed by its conjugate characterization using ultraviolet (UV) spectroscopy and high-performance liquid chromatography (HPLC). The limit of detection was 10 ng/ml with a linear working range of 10 to 10,000 ng/ml. The affinity constant (K_a) of the vitamin B₁₂ antibody was found to be 4.23 × 10⁸ L/mol. Cross-reactivity with other water-soluble vitamins was found to be less than 0.01% except for analogs of vitamin B₁₂ that showed 12% to 35%. The intra- and interassay coefficients of variation were found to be in the ranges from 0.0005% to 1.2% and 0.009% to 1.03%, respectively. The assay was validated with the HPLC method in terms of sensitivity, specificity, precision, and recovery of vitamin B₁₂ with spiked multivitamin injections, tablets, capsules, and chocolates. The HPLC method had a detection limit of 500 ng/ml with a linear working range of 1000 to 10,000 ng/ml. After extraction of vitamin B₁₂ using Amberlite XAD, the developed ELISA method correlated well with the established HPLC method with a correlation coefficient of 0.90.     

Impact of Helicobacter pylori on the development of vitamin B12 deficiency in the absence of gastric atrophy

Background. Cobalamin (vitamin B₁₂) deficiency is associated with Helicobacter pylori infection. This study examined how serum vitamin B₁₂ levels relate to gastric mucosa H. pylori density and histology, and to hematological findings in patients with minimal or no gastric atrophy. A second aim was to confirm that H. pylori eradication therapy increases serum B₁₂. Materials and Methods. Biopsies of the gastric mucosa from a population of dyspeptic patients were graded for level of chronic inflammation, neutrophil activity, atrophy, and H. pylori density. A total of 145 H. pylori‐infected patients with minimal or no atrophy were included in the study. Serum cobalamin level, hemoglobin level, and mean corpuscular volume were measured in the 145 patients before eradication therapy, and in 65 of the subjects after treatment. The hematologic findings before and after eradication therapy and correlations between serum vitamin B₁₂ level and histologic parameters, hematologic findings, and patient age were statistically analyzed. Results. There was no significant correlation between serum cobalamin level and patient age. Before treatment all the histopathological scores were inversely correlated with serum vitamin B₁₂ level (p < .01) on univariate analysis. Only H. pylori density was significantly associated with B₁₂ level on multivariate analysis. Serum hemoglobin and cobalamin levels were significantly increased after treatment, regardless of H. pylori eradication status (p < .001). Conclusion. The findings provide strong evidence that H. pylori infection is associated with cobalamin deficiency, and show that this is true even in patients with nonulcer dyspepsia and minimal or no gastric atrophy.     

Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety

In order to elucidate the biosynthesis of the base moiety of cobalamin in Salmonella typhimurium LT2, this organism was grown in the presence of [1′-¹⁴C]riboflavin. The vitamin B₁₂ isolated was ¹⁴C-labeled. It was shown by chemical degradation that the ¹⁴C-label was exclusively localized in carbon atom 2 of the 5,6-dimethylbenzimidazole moiety. This demonstrated the precursor function of riboflavin in the biosynthesis of 5,6-dimethylbenzimidazole in S. typhimurium. Received: 25 August 1998 / Accepted: 27 October 1998