Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene.

The vitamin B12 (B12)-mediated repression of the metE gene in Escherichia coli and Salmonella typhimurium requires the B12-dependent transmethylase, the metH gene product. It has been proposed that the MetH-B12 holoenzyme complex is involved directly in the repression mechanism. Using Escherichia coli strains lysogenized with a lambda phage carrying a metE-lacZ gene fusion, we examined B12-mediated repression of the metE-lacZ gene fusion. Although B12 supplementation results in a 10-fold repression of metE-lacZ expression, homocysteine addition to the growth medium overrides the B12-mediated repression. In addition, B12-mediated repression of the metE-lacZ fusion is dependent on a functional MetR protein. When a metB mutant was transformed with a high-copy-number plasmid carrying the metE gene, which would be expected to reduce intracellular levels of homocysteine, metE-lacZ expression was reduced and B12 supplementation had no further effect. In a metJ mutant, B12 represses metE-lacZ expression less than twofold. When the metJ mutant was transformed with a high-copy-number plasmid carrying the metH gene, which would be expected to reduce intracellular levels of homocysteine, B12 repression of the metE-lacZ fusion was partially restored. The results indicate that B12-mediated repression of the metE gene is primarily a loss of MetR-mediated activation due to depletion of the coactivator homocysteine, rather than a direct repression by the MetH-B12 holoenzyme.     

A spectroscopic analysis of vitamin B12 derivatives.

1. Several vitamin B12 derivatives including descobalt B12 show unusual inversion of their CD signs upon rapid cooling to liquid N2 temperature, although the room temperature CD signs are conserved by a slower cooling to the same temperature. As a possible explanation for this puzzling observation, a micro-environmental birefringence around the chromophore imbedded in an organic glass is proposed. 2. Absorption, fluorescence, phosphorescence, and polarization spectra of descobalt B12 can be correlated with those of porphyrin free bases, as these two molecular systems share many similarites in their electronic structure. 3. Molecular orbital calculations of polarization directions further support the analoby between the spectroscopic characteristics of corrins and porphyrins, and are generally in good agreement with the fluorescence polarization data.     

Repression of synthesis of the vitamin B12 receptor in Escherichia coli.

Growth of Escherichia coli K-12 strains in the presence of the vitamin cyanocobalamin (B12) resulted in an 80 to 90% reduction in B12 uptake activity of washed cells. Coincident with the decline in uptake activity was the depression of B12-binding activity in energy-poisoned cells, suggesting that growth in B12 resulted in the repression of synthesis of the B12 receptor protein in the outer membrane. Growth in the presence of B12 led to marked reduction in sensitivity to the E colicins, whose adsorption to cells requires the B12 receptor, and to a decrease in the amount of a band on electropherograms of outer membrane proteins. That polypeptide was also missing from mutants altered at btuB, the locus encoding the B12 receptor. Addition of B12 to growing cultures resulted in the exponential decline in specific activity of B12 uptake, as expected for dilution of functional receptors by further growth. Repression of receptor synthesis appears to be regulated by the level of intracellular, rather than extracellular, B12 and is separate from the regulation of the methionine biosynthetic pathway. Mutants altered in btuC, which are defective in accumulation and retention of B12, exhibit a much lower degree of repressibility.     

The biosynthesis of vitamin B12.

The use of 13C-Fourier transform nuclear magnetic resonance (F.t.-n.m.r.) has led to the observation that while 8 molecules of [2-13C]ALA are incorporated into vitamin B12 in P. shermanii, [5-13C]ALA labels only seven of the carbon atoms of cyanocobalamin, i.e. one of the amino methyl groups of ALA is "lost" in the process. It has also been confirmed that seven of the methyl groups of B12 are derived from 13CH3-enriched methionine and further that the chirality of the gemdimethyl grouping at C12 labelled with [13CH3]methionine is R. A soluble enzyme mixture from the 37000 or 100000 g supernatant of disrupted cells of P. shermanii converts both 14 C-labelled ALA and [14C]uro'gen III to cobyrinic acid, the simplest corrinoid material on the pathway to vitamin B12 and the coenzyme, in presence of NADPH, Co2+, Mg2+, S-adenosyl-methionine and glutathione. Multiply-labelled uro'gens (13C, 14C and 3H) have been used to show that incorporation takes place without randomization. A sequence for corrin synthesis from uro'gen III is presented.     

Relationships between biotin and vitamin B12. Effects of biotin and vitamin B12 on folic acid metabolism

1. The effects of dietary biotin compared with vitamin B₁₂ on the total content and on the distribution of the various folate derivatives in the liver of rats given a biotin-free diet have been studied. The effect of both vitamins on the conversion in vitro of folic acid into citrovorum factor in the same experimental conditions was also examined. 2. In biotin-treated rats as well as in vitamin B₁₂-treated rats the total content of folic acid-active substances measured microbiologically by Pediococcus cerevisiae, Streptococcus faecalis and Lactobacillus casei is significantly higher than that in biotin-deficient rats. The liver distribution of various folate derivatives in the three groups of animals is also markedly modified. 3. The amount of citrovorum factor formed in systems with liver homogenate of rats receiving biotin or vitamin B₁₂ is higher than that with liver homogenates of deficient rats. 4. The results obtained demonstrate the influence of biotin in the metabolism of folic acid, and the similar actions at this level of both biotin and vitamin B₁₂. These results are discussed in relation to the participation of the two vitamins in the metabolism of C₁ units, as a biochemical interpretation of the relationships between vitamin B₁₂ and biotin.     

A gene, cobA + hemD, from Selenomonas ruminantium encodes a bifunctional enzyme involved in the synthesis of vitamin B12.

Coenzymes derived from vitamin B12 (cyanocobalamin) are particularly important for core metabolism in ruminant animals. Selenomonas ruminantium, a Gram-positive obligate anaerobe isolated from cattle, is the main contributor of vitamin B12 to such ruminant animals. In nature, there are both aerobic and anaerobic pathways for B12 synthesis - the latter is only partly elucidated. Until now, there has been no investigation of B12 synthesis in S. ruminantium, which must use an anaerobic pathway. This paper reports the cloning of the chromosomal operon from S. ruminantium which is responsible for the first committed steps in corrinoid synthesis. Five open reading frames were found in the cloned fragment. All deduced amino acid sequences had similarity to defined proteins in the databases that are involved in porphyrin and corrin synthesis. Of particular interest is the gene designated cobA + hemD, which encodes a single polypeptide possessing two catalytic functions - uroporphyrinogen III synthase and uroporphyrinogen III 2,7-methyltransferase. This enzyme converts hydroxymethylbilane to precorrin-2. The functions of the protein coded by cobA + hemD were established by heterologous expression in Escherichia coli. The CobA activity has been demonstrated for three distinct types of proteins - monofunctional, bifunctional with siroheme formation and, this report, bifunctional with uroporphyrinogen III synthesis. The type found in S. ruminantium (cobA + hemD) is probably restricted to obligately anaerobic fermentative bacteria.