Comparative Genomics Guides Elucidation of Vitamin B12 Biosynthesis in Novel Human-Associated Akkermansia Strains.

Akkermansia muciniphila is a mucin-degrading bacterium found in the gut of most humans and is considered a "next-generation probiotic." However, knowledge of the genomic and physiological diversity of human-associated Akkermansia sp. strains is limited. Here, we reconstructed 35 metagenome-assembled genomes and combined them with 40 publicly available genomes for comparative genomic analysis. We identified at least four species-level phylogroups (AmI to AmIV), with distinct functional potentials. Most notably, we identified genes for cobalamin (vitamin B12) biosynthesis within the AmII and AmIII phylogroups. To verify these predictions, 10 Akkermansia strains were isolated from adults and screened for vitamin B12 biosynthesis genes via PCR. Two AmII strains were positive for the presence of cobalamin biosynthesis genes, while all 9 AmI strains tested were negative. To demonstrate vitamin B12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups, since cobalamin is an essential cofactor in propionate metabolism. Results showed that the AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of vitamin B12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B12 was not supplied in the culture medium. Lastly, two bioassays were used to confirm vitamin B12 production by the AmII phylogroup. This novel physiological trait of human-associated Akkermansia strains may affect how these bacteria interact with the human host and other members of the human gut microbiome.IMPORTANCE There is significant interest in the therapeutic and probiotic potential of the common gut bacterium Akkermansia muciniphila However, knowledge of both the genomic and physiological diversity of this bacterial lineage is limited. Using a combination of genomic, molecular biological, and traditional microbiological approaches, we identified at least four species-level phylogroups with differing functional potentials that affect how these bacteria interact with both their human host and other members of the human gut microbiome. Specifically, we identified and isolated Akkermansia strains that were able to synthesize vitamin B12 The ability to synthesize this important cofactor broadens the physiological capabilities of human-associated Akkermansia strains, fundamentally altering our understanding of how this important bacterial lineage may affect human health.

Biochemistry and Physiology of Vitamins in Euglena.

Euglena gracilis Z requires vitamins B1 and B12 for growth. It takes up and accumulates large amounts of these exogenous vitamins through energy-dependent active transport systems. Except for these essential vitamins, E. gracilis Z has the ability to synthesize all human vitamins. Euglena synthesizes high levels of antioxidant vitamins such as vitamins C and E, and, thus, are used as nutritional supplements for humans and domestic animals. Methods to effectively produce vitamins in Euglena have been investigated.Previous biochemical studies indicated that E. gracilis Z contains several vitamin-related novel synthetic enzymes and metabolic pathways which suggests that it is a highly suitable organism for elucidating the physiological functions of vitamins in comparative biochemistry and biological evolution. E. gracilis Z has an unusual biosynthetic pathway for vitamin C, a hybrid of the pathways found in animals and plants. This chapter presents up-to-date information on the biochemistry and physiological functions of vitamins in this organism.

Phenotypic and genotypic screening of human-originated lactobacilli for vitamin B12 production potential: process validation by micro-assay and UFLC.

Vitamin B12 (B12) production is a strain specific, rare and hidden functional attribute of lactobacilli and a cogent protocol for selection of such isolates from the herd of lactobacilli is required. The present study included isolation of lactobacilli from human samples (milk and fecal), screening them by a polyphasic (three-phase) methodology for probable B12 production potential and validating the screening protocol by exploring selected strains for in vitro vitamin production (two-phase fermentation) and quantification [micro-assay and ultra fast liquid chromatography (UFLC)]. Fifty-nine Lactobacillus strains were recovered from tested biological samples. Contrary to screening inapplicabilities of first [growth potential (GP) in B12-free medium] and second phases (GP in B12-free and cobalt chloride-supplemented conditions), third phase (cbiK gene detection on genomic DNA) alone was revealed as a validated strategy for selection of two probable B12-producing lactobacilli. Microbiological assay confirmed production and bioavailability of produced vitamin, while UFLC testing validated the results by precisely quantifying the cyanocobalamin (industrially produced bio-available form of B12) in cell extracts of both possible B12 producers [BHM10 (10.91 ± 1.55 µg/l) and BCF20 (23.90 ± 1.73 µg/l)] and positive standard [Lactobacillus reuteri DSM20016 (20.03 ± 4.17 µg/l)]. Moreover, this study generates a novel report for genomic detection, partial amplification and sequencing of cbiK gene in Lactobacillus plantarum species (both BHM10 and BCF20). In conclusion, contrary to first two phases, cbiK gene detection strategy successfully selects B12-producing strains from a group of human-originated lactobacilli and can be used in the future for similar screening studies.

Auxotrophy and intrapopulation complementary in the 'interactome' of a cultivated freshwater model community.

Microorganisms are usually studied either in highly complex natural communities or in isolation as monoclonal model populations that we manage to grow in the laboratory. Here, we uncover the biology of some of the most common and yet-uncultured bacteria in freshwater environments using a mixed culture from Lake Grosse Fuchskuhle. From a single shotgun metagenome of a freshwater mixed culture of low complexity, we recovered four high-quality metagenome-assembled genomes (MAGs) for metabolic reconstruction. This analysis revealed the metabolic interconnectedness and niche partitioning of these naturally dominant bacteria. In particular, vitamin- and amino acid biosynthetic pathways were distributed unequally with a member of Crenarchaeota most likely being the sole producer of vitamin B12 in the mixed culture. Using coverage-based partitioning of the genes recovered from a single MAG intrapopulation metabolic complementarity was revealed pointing to 'social' interactions for the common good of populations dominating freshwater plankton. As such, our MAGs highlight the power of mixed cultures to extract naturally occurring 'interactomes' and to overcome our inability to isolate and grow the microbes dominating in nature.

Vitamin B12 modulates the transcriptome of the skin microbiota in acne pathogenesis.

Various diseases have been linked to the human microbiota, but the underlying molecular mechanisms of the microbiota in disease pathogenesis are often poorly understood. Using acne as a disease model, we aimed to understand the molecular response of the skin microbiota to host metabolite signaling in disease pathogenesis. Metatranscriptomic analysis revealed that the transcriptional profiles of the skin microbiota separated acne patients from healthy individuals. The vitamin B12 biosynthesis pathway in the skin bacterium Propionibacterium acnes was significantly down-regulated in acne patients. We hypothesized that host vitamin B12 modulates the activities of the skin microbiota and contributes to acne pathogenesis. To test this hypothesis, we analyzed the skin microbiota in healthy subjects supplemented with vitamin B12. We found that the supplementation repressed the expression of vitamin B12 biosynthesis genes in P. acnes and altered the transcriptome of the skin microbiota. One of the 10 subjects studied developed acne 1 week after vitamin B12 supplementation. To further understand the molecular mechanism, we revealed that vitamin B12 supplementation in P. acnes cultures promoted the production of porphyrins, which have been shown to induce inflammation in acne. Our findings suggest a new bacterial pathogenesis pathway in acne and provide one molecular explanation for the long-standing clinical observation that vitamin B12 supplementation leads to acne development in a subset of individuals. Our study discovered that vitamin B12, an essential nutrient in humans, modulates the transcriptional activities of skin bacteria, and provided evidence that metabolite-mediated interactions between the host and the skin microbiota play essential roles in disease development.

Enhancing vitamin B12 content in soy-yogurt by Lactobacillus reuteri.

More attention from the aged and vegetarians has been paid to soy-product due to its taste, easy digestibility, as well as the association with health. However, soy-product has a defect of low vitamin content, mainly the water-soluble vitamin B12. This study was to investigate co-fermentation of glycerol and fructose in soy-yogurt to enhance vitamin B12 production by Lactobacillus reuteri. After a serial combination experiments, the co-fermentation was confirmed to enhance the production of vitamin B12 up to 18 µg/100mL. Both supplementations induced the expression of cobT and cbiA and functioned to balance the redox reaction. Meanwhile, high content of fructose supplementation reduced the production of vitamin B12 and suppressed expression of cobT in bacteria. It was proved that the vitamin B12 content of this soy-yogurt is higher than other fermented soybean based food and thus can be served as an alternative food for the aged and vegetarians.

Fundamental role of cobalamin biosynthesis in the developmental growth of Streptomyces coelicolor A3 (2).

Cobalamin (Cbl) (synonym, vitamin B12) is the cobalt-containing cofactor produced only by some prokaryotes. Streptomyces is an effective Cbl producer. To study the role of Cbl production in Streptomyces, a knockout mutant for Cbl biosynthesis (cob) was generated in Streptomyces coelicolor A3 (2). The growth of the mutant was similar to that of the wild type in a rich medium, but inhibited in minimal medium, suggesting the involvement of Cbl in some step of primary metabolism. Methionine synthesis catalyzed by MetH, the Cbl-dependent methionine synthase, is a candidate. However, supplementing the minimal medium with methionine did not rescue the growth of the cob mutant, indicating that the availability of Cbl affects another primary function. Transcriptional analysis confirmed that the mutant induced metE encoding an alternative Cbl-independent methionine synthase, probably due to the Cbl-dependent riboswitch mechanism. The cob mutant produced low levels of pigment antibiotics and formed fewer aerial mycelium and spores in a rich medium, suggesting that a Cbl-dependent mechanism controls development. A similar developmental defect was observed for a knockout mutant for SCO4800, encoding the putative Cbl-dependent isobutyryl-CoA mutase (Icm) small subunit. Since the knockout of the Icm large subunit (SCO5415) did not affect the developmental phenotype, SCO4800 likely regulates development independently from SCO5415. Effective Cbl production is fundamental to the diverse functions underlying the complex developmental life cycle of S. coelicolor A3 (2).

Development of a two-step cultivation strategy for the production of vitamin B12 by Bacillus megaterium.

BACKGROUND: Vitamin B12 is a fascinating molecule which acts as a co-factor in the metabolism of many organisms, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production. The synthesis of vitamin B12 is limited to a few of bacteria and archaea. Therefore, industrial microbial fermentation is used to meet annual demands worldwide of vitamin B12 and as an alternative method to the chemical synthesis which requires at least 60 steps that is uneconomical. Bacillus megaterium is one of vitamin B12 producers and an ideal host for many biotechnology applications and being one of the best tools for the industrial production of several enzymes. Therefore, a two-step optimization strategy was established to produce high yield of vitamin B12 by B. megaterium through the provision of the production requirements and the suitable conditions for the biosynthesis of vitamin B12. RESULTS: We achieved the optimum conditions for the fermentation process of B. megaterium to produce high yield of vitamin B12 in a practical way based on statistical design and analysis which allowed vitamin B12 production to increase up to 759-fold (204.46 µg/l) as compared with control without parameters (0.26 µg/L). High performance liquid chromatography coupled to variable wavelength detector and mass spectrometry has been used to identify vitamin B12 forms and confirm the results. CONCLUSIONS: We developed the fermentation process of B. megaterium to enhance the production of vitamin B12 by providing the required supplements for the synthesis of vitamin B12 (CoCl2, δ-aminolevulinic acid (ALA) and 5,6-dimethylbenzimidazole (DMB)) and dividing the fermentation process into three stages. In addition, the optimum incubation times of the three fermentation stages were investigated and performed with reducing number of experimental and evaluated multiple parameters and their interactions by using statistical experimental design and analysis. All of these strategies has proven successful in enhancing the production of vitamin B12 up to 204.46 µg/l and demonstrated that B. megaterium could be a good candidate for the industrial production of vitamin B12.

Towards a cell factory for vitamin B12 production in Bacillus megaterium: bypassing of the cobalamin riboswitch control elements.

Bacillus megaterium is a bacterium that has been used in the past for the industrial production of vitamin B12 (cobalamin), the anti-pernicious anaemia factor. Cobalamin is a modified tetrapyrrole with a cobalt ion coordinated within its macrocycle. More recently, B. megaterium has been developed as a host for the high-yield production of recombinant proteins using a xylose inducible promoter system. Herein, we revisit cobalamin production in B. megaterium DSM319. We have investigated the importance of cobalt for optimum growth and cobalamin production. The cobaltochelatase (CbiX(L)) is encoded within a 14-gene cobalamin biosynthetic (cbi) operon, whose gene-products oversee the transformation of uroporphyrinogen III into adenosylcobyrinic acid a,c-diamide, a key precursor of cobalamin synthesis. The production of CbiX(L) in response to exogenous cobalt was monitored. The metal was found to stimulate cobalamin biosynthesis and decrease the levels of CbiX(L). From this we were able to show that the entire cbi operon is transcriptionally regulated by a B12-riboswitch, with a switch-off point at approximately 5 nM cobalamin. To bypass the effects of the B12-riboswitch the cbi operon was cloned without these regulatory elements. Growth of these strains on minimal media supplemented with glycerol as a carbon source resulted in significant increases in cobalamin production (up to 200 µg L(-1)). In addition, a range of partially amidated intermediates up to adenosylcobyric acid was detected. These findings outline a potential way to develop B. megaterium as a cell factory for cobalamin production using cheap raw materials.

Thermotoga lettingae can salvage cobinamide to synthesize vitamin B12.

We recently reported that the Thermotogales acquired the ability to synthesize vitamin B12 by acquisition of genes from two distantly related lineages, Archaea and Firmicutes (K. S. Swithers et al., Genome Biol. Evol. 4:730-739, 2012). Ancestral state reconstruction suggested that the cobinamide salvage gene cluster was present in the Thermotogales' most recent common ancestor. We also predicted that Thermotoga lettingae could not synthesize B12 de novo but could use the cobinamide salvage pathway to synthesize B12. In this study, these hypotheses were tested, and we found that Tt. lettingae did not synthesize B12 de novo but salvaged cobinamide. The growth rate of Tt. lettingae increased with the addition of B12 or cobinamide to its medium. It synthesized B12 when the medium was supplemented with cobinamide, and no B12 was detected in cells grown on cobinamide-deficient medium. Upstream of the cobinamide salvage genes is a putative B12 riboswitch. In other organisms, B12 riboswitches allow for higher transcriptional activity in the absence of B12. When Tt. lettingae was grown with no B12, the salvage genes were upregulated compared to cells grown with B12 or cobinamide. Another gene cluster with a putative B12 riboswitch upstream is the btuFCD ABC transporter, and it showed a transcription pattern similar to that of the cobinamide salvage genes. The BtuF proteins from species that can and cannot salvage cobinamides were shown in vitro to bind both B12 and cobinamide. These results suggest that Thermotogales species can use the BtuFCD transporter to import both B12 and cobinamide, even if they cannot salvage cobinamide.

Organic cofactors in the metabolism of Dehalococcoides mccartyi strains.

Dehalococcoides mccartyi strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published Dehalococcoides genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of Dehalococcoides physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in Dehalococcoides strain CBDB1. Dehalococcoides genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B12), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole. Dehalococcoides genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate, S-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes.

Genomic determinants of organohalide-respiration in Geobacter lovleyi, an unusual member of the Geobacteraceae.

BACKGROUND: Geobacter lovleyi is a unique member of the Geobacteraceae because strains of this species share the ability to couple tetrachloroethene (PCE) reductive dechlorination to cis-1,2-dichloroethene (cis-DCE) with energy conservation and growth (i.e., organohalide respiration). Strain SZ also reduces U(VI) to U(IV) and contributes to uranium immobilization, making G. lovleyi relevant for bioremediation at sites impacted with chlorinated ethenes and radionuclides. G. lovleyi is the only fully sequenced representative of this distinct Geobacter clade, and comparative genome analyses identified genetic elements associated with organohalide respiration and elucidated genome features that distinguish strain SZ from other members of the Geobacteraceae. RESULTS: Sequencing the G. lovleyi strain SZ genome revealed a 3.9 Mbp chromosome with 54.7% GC content (i.e., the percent of the total guanines (Gs) and cytosines (Cs) among the four bases within the genome), and average amino acid identities of 53-56% compared to other sequenced Geobacter spp. Sequencing also revealed the presence of a 77 kbp plasmid, pSZ77 (53.0% GC), with nearly half of its encoded genes corresponding to chromosomal homologs in other Geobacteraceae genomes. Among these chromosome-derived features, pSZ77 encodes 15 out of the 24 genes required for de novo cobalamin biosynthesis, a required cofactor for organohalide respiration. A plasmid with 99% sequence identity to pSZ77 was subsequently detected in the PCE-dechlorinating G. lovleyi strain KB-1 present in the PCE-to-ethene-dechlorinating consortium KB-1. Additional PCE-to-cis-DCE-dechlorinating G. lovleyi strains obtained from the PCE-contaminated Fort Lewis, WA, site did not carry a plasmid indicating that pSZ77 is not a requirement (marker) for PCE respiration within this species. Chromosomal genomic islands found within the G. lovleyi strain SZ genome encode two reductive dehalogenase (RDase) homologs and a putative conjugative pilus system. Despite the loss of many c-type cytochrome and oxidative-stress-responsive genes, strain SZ retained the majority of Geobacter core metabolic capabilities, including U(VI) respiration. CONCLUSIONS: Gene acquisitions have expanded strain SZ's respiratory capabilities to include PCE and TCE as electron acceptors. Respiratory processes core to the Geobacter genus, such as metal reduction, were retained despite a substantially reduced number of c-type cytochrome genes. pSZ77 is stably maintained within its host strains SZ and KB-1, likely because the replicon carries essential genes including genes involved in cobalamin biosynthesis and possibly corrinoid transport. Lateral acquisition of the plasmid replicon and the RDase genomic island represent unique genome features of the PCE-respiring G. lovleyi strains SZ and KB-1, and at least the latter signifies adaptation to PCE contamination.

B-group vitamin production by lactic acid bacteria--current knowledge and potential applications.

Although most vitamins are present in a variety of foods, human vitamin deficiencies still occur in many countries, mainly because of malnutrition not only as a result of insufficient food intake but also because of unbalanced diets. Even though most lactic acid bacteria (LAB) are auxotrophic for several vitamins, it is now known that certain strains have the capability to synthesize water-soluble vitamins such as those included in the B-group (folates, riboflavin and vitamin B(12) amongst others). This review article will show the current knowledge of vitamin biosynthesis by LAB and show how the proper selection of starter cultures and probiotic strains could be useful in preventing clinical and subclinical vitamin deficiencies. Here, several examples will be presented where vitamin-producing LAB led to the elaboration of novel fermented foods with increased and bioavailable vitamins. In addition, the use of genetic engineering strategies to increase vitamin production or to create novel vitamin-producing strains will also be discussed. This review will show that the use of vitamin-producing LAB could be a cost-effective alternative to current vitamin fortification programmes and be useful in the elaboration of novel vitamin-enriched products.

Convergent evolution of metabolic roles in bacterial co-symbionts of insects.

A strictly host-dependent lifestyle has profound evolutionary consequences for bacterial genomes. Most prominent is a sometimes-dramatic amount of gene loss and genome reduction. Recently, highly reduced genomes from the co-resident intracellular symbionts of sharpshooters were shown to exhibit a striking level of metabolic interdependence. One symbiont, called Sulcia muelleri (Bacteroidetes), can produce eight of the 10 essential amino acids, despite having a genome of only 245 kb. The other, Baumannia cicadellinicola (gamma-Proteobacteria), can produce the remaining two essential amino acids as well as many vitamins. Cicadas also contain the symbiont Sulcia, but lack Baumannia and instead contain the co-resident symbiont Hodgkinia cicadicola (alpha-Proteobacteria). Here we report that, despite at least 200 million years of divergence, the two Sulcia genomes have nearly identical gene content and gene order. Additionally, we show that despite being phylogenetically distant and drastically different in genome size and architecture, Hodgkinia and Baumannia have converged on gene sets conferring similar capabilities for essential amino acid biosynthesis, in both cases precisely complementary to the pathways conserved in Sulcia. In contrast, they have completely divergent capabilities for vitamin biosynthesis. Despite having the smallest gene set known in bacteria, Hodgkinia devotes at least 7% of its proteome to cobalamin (vitamin B(12)) biosynthesis, a significant metabolic burden. The presence of these genes can be explained by Hodgkinia's retention of the cobalamin-dependent version of methionine synthase instead of the cobalamin-independent version found in Baumannia, a situation that necessitates retention of cobalamin biosynthetic capabilities to make the essential amino acid methionine.

Bacterial vitamin B2, B11 and B12 overproduction: An overview.

Consumers are becoming increasingly health conscious and therefore more discerning in their food choices. The production of fermented food products with elevated levels of B-vitamins increase both their commercial and nutritional value, and eliminate the need for subsequent fortification with these essential vitamins. Such novel products could reduce the incidence of inadequate vitamin intake which is common in many parts of the world, not only in developing countries, but also in many industrialised countries. Moreover, the concept of in situ fortification by bacterial fermentation opens the way for development of food products targeted at specific groups in society such as the elderly and adolescents. This review looks at how vitamin overproduction strategies have been developed, some of which have successfully been tested in animal models. Such innovative strategies could be relatively easily adapted by the food industry to develop novel vitamin-enhanced functional foods with enhanced consumer appeal.

The influence of an increased cobalt supply on ruminal parameters and microbial vitamin B12 synthesis in the rumen of dairy cows.

The aim of the study was to examine the effects of an elevated dietary cobalt supply to dairy cows on rumen fermentation parameters and microbial vitamin B12 synthesis in the rumen. Five lactating dairy cows fitted with a ruminal and a duodenal cannula were subsequently fed either a ration containing only the native cobalt content (0.17 mg Co/ kg DM) or a ration supplemented with cobalt sulphate (0.29 mg Co/kg DM). The pH-value, the ammonia concentration as well as the concentration and the molar proportions of short chain fatty acids in the rumen were not significantly influenced by feeding the ration with the higher cobalt content. While there was no difference in microbial protein flow, the cobalamin flow at the duodenum was significantly elevated in supplemented animals (3.67 +/- 0.69 vs. 8.63 +/- 2.22 mg B12/d). The efficiency of cobalt utilisation for ruminal vitamin B12 synthesis was calculated to be 7.1 +/- 1.3% for the unsupplemented and 9.5 +/- 2.4% for the supplemented ration. Further investigation has to prove if there are any benefits for cows resulting from the elevated cobalamin synthesis measured, caused by feeding higher amounts of dietary cobalt.

Identification and quantitation of cobalamin and cobalamin analogues in human feces.

BACKGROUND: Cobalamin (vitamin B-12) and cobalamin analogues are present in human feces, but a complete identification has not been established, and the amounts present have not been determined. OBJECTIVES: We aimed to develop a liquid chromatography-mass spectrometry method for cobalamin and cobalamin analogues and to identify and quantitiate the amounts present in human feces. DESIGN: Fecal samples were obtained from 20 human subjects in good general health. The samples were analyzed for the presence and amounts of cobalamin and 12 cobalamin analogues that were synthesized with and without the incorporation of stable isotopes. RESULTS: Cobalamin and 7 cobalamin analogues were identified and quantitated in human feces. The mean for the total amount present in 18 subjects whose daily intake was < or = 25 microg cobalamin from vitamin supplements was 1309 ng cobalamin equivalents/g wet wt of feces. Cobalamin (1.4%) and cobinamide (1.8%) (an incomplete corrinoid) represented a small portion of the total amount. Six cobalamin analogues that contain a base other than the 5,6-dimethylbenzimidizidole in cobalamin were present. The bases and their mean amounts (in %) are 2-methyladenine (60.6%), p-cresol (16.3%), adenine (12.5%), 2-(methylthio)adenine (15.5%), 5-hydroxybenzimidazole (1.8%), and phenol (0.1%). One subject ingested 1 mg cobalamin/d and another ingested 2 mg cobalamin/d, and they appeared to convert most of the cobalamin to cobinamide and the 4 analogues that contain the bases-2-methyladenine, p-cresol, adenine, and 2-(methylthio)adenine. CONCLUSIONS: Cobalamin analogues are present in human feces and account for > 98% of the total of cobalamin plus cobalamin analogues. A major portion of large amounts of ingested cobalamin appears to be converted to cobalamin analogues.

Enhanced anaerobic biotransformation of carbon tetrachloride with precursors of vitamin B(12) biosynthesis.

Relatively low concentrations of Vitamin B(12) are known to accelerate the anaerobic biotransformation of carbon tetrachloride (CT) and chloroform (CF). However, the addition of vitamin B(12) for field-scale bioremediation is expected to be costly. The present study considered a strategy to generate vitamin B(12) by addition of biosynthetic precursors. One of the precursors, porphobilinogen (PB) involved in the formation of the corrin ring, significantly increased the CT biotransformation rates by 2.7-, 8.8- and 10.9-fold when supplemented at 160, 500 and 900 microM, respectively. A positive control with 10 microM of vitamin B(12) resulted in a 5.9-fold increase in the CT-bioconversion rate. PB additions provided high molar yields of inorganic chloride (57% of CT organochlorine), comparable to that obtained with vitamin B(12) supplemented cultures. The primary substrate, methanol, known to induce vitamin B(12) production in methanogens and acetogens, was required for PB to have a significant impact on CT conversion. The observation suggests that PB's role was due to stimulating vitamin B(12) biosynthesis. The present study therefore provides insights on how to achieve vitamin B(12) enhanced rates of CT bioremediation through the use of less complex compounds that are precursors of vitamin B(12). Although PB is a costly chemical, its large impact points to corrin ring formation as the rate-limiting step.

Influence of cobalt concentration on vitamin B12 production and fermentation of mixed ruminal microorganisms grown in continuous culture flow-through fermentors.

An experiment was conducted to determine the effects of dietary concentrations of Co on vitamin B12 production and fermentation of mixed ruminal microbes grown in continuous culture fermentors. Four fermentors were fed 14 g of DM/d. The DM consisted of a corn and cottonseed hull-based diet with Co supplemented as CoCO3. Dietary treatments were 1) control (containing 0.05 mg of Co/kg of DM), 2) 0.05 mg of supplemental Co/kg of DM, 3) 0.10 mg of supplemental Co/kg of DM, and 4) 1.0 mg of supplemental Co/kg of DM. After a 3-d adjustment period, fermentors were sampled over a 3-d sampling period. This process was repeated 2 additional times for a total of 3 runs. Ruminal fluid vitamin B12 concentrations were affected by Co supplementation (P < 0.01), and there was a treatment x day interaction (P < 0.01). By sampling d 3, cultures fed the basal diet supplemented with 0.10 mg of Co/kg had greater (P < 0.05) vitamin B12 concentrations than those supplemented with 0.05 mg of Co/kg of DM, and increasing supplemental Co from 0.10 to 1.0 mg/kg of DM increased (P < 0.01) ruminal fluid vitamin B12 concentration. Ruminal fluid succinate also was affected (P < 0.10) by a treatment x day interaction. Cobalt supplementation to the control diet greatly decreased (P < 0.05) succinate in ruminal cultures on sampling d 3 but not on d 1 or 2. Molar proportions of acetate, propionate, and isobutyrate, and acetate:propionate were not affected by the addition of supplemental Co to the basal diet. However, molar proportions of butyrate, valerate, and isovalerate increased (P < 0.05) in response to supplemental Co. The majority of long-chain fatty acids observed in this study were not affected by Co supplementation. However, percentages of C18:0 fatty acids in ruminal cultures tended (P < 0.10) to be greater for Co-supplemented diets relative to the control. Methane, ammonia, and pH were not greatly affected by Co supplementation. The results indicate that a total (diet plus supplemental) Co concentration of 0.10 to 0.15 mg/kg of dietary DM resulted in adequate vitamin B12 production to meet the requirements of ruminal microorganisms fed a high-concentrate diet in continuous-flow fermentors.

Dietary forage and nonfiber carbohydrate contents influence B-vitamin intake, duodenal flow, and apparent ruminal synthesis in lactating dairy cows.

The objective of this experiment was to quantify intakes, duodenal flows, and ruminal apparent synthesis (AS) of B-vitamins in lactating dairy cows fed diets varying in forage and nonfiber carbohydrate (NFC) contents. Eight (4 primiparous and 4 multiparous) ruminally and duodenally cannulated Holstein cows were assigned to 4 dietary treatments in a replicated 21-d period, 4 x 4 Latin square design with a 2 x 2 factorial treatment arrangement. Diets, fed as TMR, contained (DM basis) 2 levels of forage (35 and 60%) and 2 levels of NFC (30 and 40%). The forage portion of the diets contained 50% corn silage, 33% alfalfa hay, and 17% grass hay. Soybean hulls and beet pulp (2:1) and corn meal and ground barley (2:1) were included to achieve desired NFC concentrations. No supplemental B-vitamins were fed. B-vitamin AS was calculated as the amount of a specific B-vitamin flowing to the duodenum minus its daily orts-corrected intake. Dry matter and organic matter intakes were higher for cows fed the 35% forage diets and the 40% NFC diets. Increasing dietary forage content decreased ruminal AS of pyridoxine, folic acid, and B12. Increasing dietary NFC content increased ruminal AS of nicotinic acid, nicotinamide, niacin, pyridoxal, B6, and folic acid but decreased AS of B12. Across diets, amounts of B-vitamins synthesized were highest for niacin, followed by riboflavin, B12, thiamin, B6, and folic acid. Biotin AS values were negative for all diets, suggesting either no ruminal synthesis or that destruction by ruminal microflora was greater than synthesis. B-vitamin intake, duodenal flow, and ruminal synthesis are influenced by dietary forage and NFC contents.