Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium.

Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic conditions. Of the 30 cobalamin synthetic genes, 25 are clustered in one operon, cob, and are arranged in three groups, each group encoding enzymes for a biochemically distinct portion of the biosynthetic pathway. We have determined the DNA sequence for the promoter region and the proximal 17.1 kb of the cob operon. This sequence includes 20 translationally coupled genes that encode the enzymes involved in parts I and III of the cobalamin biosynthetic pathway. A comparison of these genes with the cobalamin synthetic genes from Pseudomonas denitrificans allows assignment of likely functions to 12 of the 20 sequenced Salmonella genes. Three additional Salmonella genes encode proteins likely to be involved in the transport of cobalt, a component of vitamin B12. However, not all Salmonella and Pseudomonas cobalamin synthetic genes have apparent homologs in the other species. These differences suggest that the cobalamin biosynthetic pathways differ between the two organisms. The evolution of these genes and their chromosomal positions is discussed.     

Molecular cloning of menaquinone biosynthetic genes of Escherichia coli K12

Summary A tranducing phage carrying some of the genes (men) defining the early stages of menaquinone biosynthesis was isolated from a pool of recombinant lambda phages that had been constructed from R.HindIII digests of E. coli DNA and the corresponding insertion vector. The lesions of menB and menC mutants were complemented by the phage but menD mutants were transduced either at low frequencies or not at all. This indicates that the transducing phage contains functional menB and menC genes but that only part of the menD gene had been cloned. The phage (G68) was accordingly disignated menCB(D). Studies with the transducing phage enabled earlier mapping data (Guest 1979) to be reinterpreted in favour of the gene order nalA.... menC..menB..menD.... purF. Restriction analyses established the presence of a bacterial DNA fragment (11.5 kb) linked by a R.HindIII target to the right arm of the genome but fused to the left arm of the vector. Hybridization studies confirmed that the cloned DNA was derived from a larger R.HindIII fragment (21 kb). A physical map of the men region was constructed and some flanking and overlapping fragments were identified.     

The CobII and CobIII regions of the cobalamin (vitamin B12) biosynthetic operon of Salmonella typhimurium.

A detailed deletion map of the CobII and CobIII regions of the cobalamin biosynthetic (cob) operon of Salmonella typhimurium LT2 has been constructed. The CobII region encodes functions needed for the synthesis of lower ligand 5,6-dimethylbenzimidazole (DMB); CobIII encodes functions needed for the synthesis of the nucleotide loop that joins DMB to the corrin macrocycle. The genetic analysis of 117 deletion, insertion, and point mutations indicates that (i) the CobII and CobIII mutations are contiguous--that is, they are grouped according to function; (ii) the CobII region is composed of four complementation groups (cobJKLM); (iii) cobM mutations do not complement mutations in any of the other three CobII groups; and (iv) CobIII mutations include three complementation groups that correspond to the cobU, cobS, and cobT genes.     

Cloning of multiple genes involved with cobalamin (Vitamin B12) biosynthesis in Bacillus megaterium.

An effective shotgun cloning procedure was developed for Bacillus megaterium by amplifying gene libraries in Bacillus subtilis. This technique was useful in isolating at least 11 genes from B. megaterium which are involved with cobalamin (vitamin B12) biosynthesis. Amplified plasmid banks were transformed into protoplasts of both a series of Cob mutants blocked before the biosynthesis of cobinamide and Cbl mutants blocked in the conversion of cobinamide into cobalamin. Amplification of gene libraries overcame the cloning barriers inherent in the relatively low protoplast transformation frequency of B. megaterium. A family of plasmids was isolated by complementation of seven different Cob and Cbl mutants. Each plasmid capable of complementing a Cob or Cbl mutant was transformed into each one of the series of Cob and Cbl mutants; many of the plasmids isolated by complementation of one mutation carried genetic activity for complementation of other mutations. By these criteria, four different complementation groups were resolved. At least six genes involved in the biosynthesis of cobinamide are carried on a fragment of DNA approximately 2.7 kilobase pairs in length; other genes involved in the biosynthesis of cobinamide were located in two other complementation groups. The physical and genetic data permitted an ordering of genes within several of the complementation groups. The presence of complementing plasmids in mutants blocked in cobalamin synthesis resulted in restoration of cobalamin biosynthesis.     

Vitamin B12 (cobalamin) deficiency in elderly patients

VITAMIN B₁₂ OR COBALAMIN DEFICIENCY occurs frequently (> 20%) among elderly people, but it is often unrecognized because the clinical manifestations are subtle; they are also potentially serious, particularly from a neuropsychiatric and hematological perspective. Causes of the deficiency include, most frequently, food-cobalamin malabsorption syndrome (> 60% of all cases), pernicious anemia (15%–20% of all cases), insufficent dietary intake and malabsorption. Food-cobalamin malabsorption, which has only recently been identified as a significant cause of cobalamin deficiency among elderly people, is characterized by the inability to release cobalamin from food or a deficiency of intestinal cobalamin transport proteins or both. We review the epidemiology and causes of cobalamin deficiency in elderly people, with an emphasis on food-cobalamin malabsorption syndrome. We also review diagnostic and management strategies for cobalamin deficiency. Vitamin B₁₂ or cobalamin deficiency occurs frequently among elderly patients,¹ but it is often unrecognized or not investigated because the clinical manifestations are subtle. However, the potential seriousness of the complications (particularly neuropsychiatric and hematological)^1,2,3,4 requires investigation of all patients who present with vitamin or nutritional deficiency. We summarize the current state of knowledge on cobalamin deficiency, with a particular focus on deficiency in elderly people.In gathering information for this article, we systematically searched PubMed for articles published from 1990 to July 2003 (the search strategy is outlined in online Appendix 1 [www.cmaj.ca/cgi/content/full/171/3/251/DC1]). We have also included unpublished data from our working group, the Groupe d''étude des carences en vitamine B12 des Hôpitaux Universitaires de Strasbourg.     

The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and propane-1,2-diol.

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably alpha 2 beta 2 gamma 2. When (R)- and (S)-propane-1,2-diols were used independently as substrates, the rate with the (R)-enantiomer was 2.5 times faster than that with the (S)-isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)-isomer was essentially the same or only slightly higher than that for the (R)-isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane-1,2-diol was determined at 2.1 A resolution. The enzyme exists as a dimer of the alpha beta gamma heterotrimer. Cobalamin is bound at the interface between the alpha and beta subunits in the so-called 'base-on' mode with 5,6-dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X-ray irradiation. The active site is in a (beta/alpha)8 barrel that was formed by a central region of the alpha subunit. The substrate propane-1,2-diol and essential cofactor K+ are bound inside the (beta/alpha)8 barrel above the corrin ring of cobalamin. K+ is hepta-coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active-site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the alpha and beta subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane-1,2-diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)-isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.     

Coexpression, purification and characterization of the E and S subunits of coenzyme B(12) and B(6) dependent Clostridium sticklandii D-ornithine aminomutase in Escherichia coli.

D-Ornithine aminomutase from Clostridium sticklandii comprises two strongly associating subunits, OraS and OraE, with molecular masses of 12,800 and 82,900 Da. Previous studies have shown that in Escherichia coli the recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies. Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate (PLP) or adenosylcobalamin (AdoCbl) play important roles in the refolding process. In this study, the DNA fragment containing both genes was cloned into the same expression vector and coexpression of the oraE and oraS genes was carried out in E. coli. The solubility of the coexpressed OraS and OraE increases with decreasing isopropyl thio-beta-D-galactoside induction temperature. Among substrate analogues tested, only 2,4-diamino-n-butyric acid displays competitive inhibition of the enzyme with a K(i) of 96 +/- 14 microm. Lys629 is responsible for the binding of PLP. The apparent K(d) for coenzyme B(6) binding to d-ornithine aminomutase is 224 +/- 41 nm as measured by equilibrium dialysis. The mutant protein, OraSE-K629M, is successfully expressed. It is catalytically inactive and unable to bind PLP. Because no coenzyme is involved in protein folding during in vivo translation of OraSE-K629M in E. coli, in vitro refolding of the enzyme employs a different folding mechanism. In both cases, the association of the S and E subunit is important for D-ornithine aminomutase to maintain an active conformation.     

Biosynthesis of cobalamin (vitamin B(12))

The biosynthesis of vitamin B(12) is summarized, emphasizing the differences observed between the aerobic and anaerobic pathways. The biosynthetic route to adenosylcobalamin from its five-carbon precursor, 5-aminolaevulinic acid, can be divided into three sections: (1) the biosynthesis of uroporphyrinogen III from 5-aminolaevulinic acid, which is common to both pathways; (2) the conversion of uroporphyrinogen III into the ring-contracted, deacylated intermediate precorrin 6 or cobalt-precorrin 6, which includes the primary differences between the two pathways; and (3) the transformation of this intermediate to form adenosylcobalamin.     

Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lysae of Escherichia coli

1. The 120-fold purification of ethanolamine ammonia-lyase from Escherichia coli extracts, to apparent homogeneity, is described. Ethanolamine, dithiothreitol, glycerol and KCl protected the apoenzyme from inactivation. 2. At the optimum pH7.5, K(m) values for ethanolamine and coenzyme B(12) were 44mum and 0.42mum respectively. The K(m) for ethanolamine was markedly affected by pH, transitions occurring at pH7.0 and 8.35. 3. The enzyme was specific for ethanolamine as substrate, none of the 18 analogues tested being active. l-2-Aminopropan-l-ol (K(i) 0.86mum), dl-1-aminopropan-2-ol (K(i) 2.2mum) and dl-1,3-diaminopropan-2-ol (K(i) 88.0mum) inhibited competitively. 4. Enzyme activity was inhibited, irreversibly and non-competitively, by the coenzyme analogues methylcobalamin (K(i) 1.4nm), hydroxocobalamin (K(i) 2.1nm) and cyanocobalamin (K(i) 4.8nm). 5. Iodoacetamide inhibited in the absence of ethanolamine, but only slightly in its presence. p-Hydroxymercuribenzoate inhibited markedly even in the presence of ethanolamine. Dithiothreitol and 2-mercaptoethanol (less effectively) restored activity to the enzyme dialysed against buffer containing ethanolamine. 6. Although K(+) ions stabilized the enzyme during dialysis or storage, they were not necessary for activity. 7. Gel filtration showed the enzyme to be of high molecular weight, ultracentrifugal studies giving s(20,w) of 16.4 and an estimated mol.wt. 560400. The isoelectric point for the apoenzyme was approx. pH5.0. inhibited enzyme activity at concentrations above 1m (95% inhibition at 3m) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated protein subunits of mol.wt. 61400. 8. Immunological studies showed that the E.coli enzyme was closely related to those of other enterobacteria, but only distantly to that of Clostridium sp. A double precipitin band suggested that the apoenzyme may be made up of two protein components.     

Increased loss of duplicated genes in streptomycin-resistant (strA) mutants of Escherichia coli k-12.

The recombination-dependent loss of a duplicated portion of the Escherichia coli chromosome is five- to tenfold greater in strains containing streptomycin resistance (strA) mutations than in the strA+ parental strain. Streptomycin (500 mug/ml) partially reverses the increase. These results suggest an interaction between strA mutations and recombination.     

Further mapping of several membrane lipid biosynthetic genes (fabC, fabB, gpsA, plsB) of Escherichia coli.

New genetic mapping data on several loci involved in membrane lipid synthesis are reported. We demonstrated that the lesion designated fabC by Broekman and Hoeckstra (Mol. Gen. Genet. 124:65-67, 1975) is a fabB mutation. In the course of this work, the orientation of the pdxB and fabB loci was determined. The order of the loci is fabB pdxB purF. We also report cotransduction between the gpsA and cysE loci and show that the order of these markers is mtl cysE gpsA. Cotransduction between the plsB and kdgK loci was also sought. Despite extensive experiments, we were unable to detect cotransduction between these loci. In addition, we were unable to detect cotransduction among several markers in region 46 to 48 min of the map.     

Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12.

The rfa locus of Escherichia coli K-12 includes a block of about 10 closely spaced genes transcribed in the same direction which are involved in synthesis and modification of the hexose region of the lipopolysaccharide core. We have sequenced the first three genes in this block. The function of the first of these genes is unknown, but we have designated it rfaQ on the basis of its location and similarity to other rfa genes. Complementation of Salmonella typhimurium rfa mutants with E. coli rfa restriction fragments indicated that the second and third genes in the block were rfaG and rfaP. The deduced sizes of the RfaQ, RfaG, and RfaP proteins are 36,298, 42,284, and 30,872 Da, respectively, and the proteins are basic and lack extensive hydrophobic domains. RfaQ shares regions of homology with proteins RfaC and RfaF, which are involved in synthesis of the heptose region of the core. Proteins RfaB, RfaG, and RfaK share a region of homology, which suggests that they belong to a second family of Rfa proteins which are thought to be hexose transferases.     

Acetaldehyde coenzyme A dehydrogenase of Escherichia coli.

Mutants of Escherichia coli (adh) in which alcohol dehydrogenase is derepressed under aerobic conditions were also found to overproduce acetaldehyde coenzyme a dehydrogenase. However, acetaldehyde coenzyme A dehydrogenase was induced by ethanol or acetaldehyde and subject to strong catabolite repression, whereas alcohol dehydrogenase was little affected by these conditions. Mutants no longer able to use ethanol as carbon source were isolated from an adh strain. Some of these mutants were revertants at the adh locus and no longer produced either alcohol dehydrogenase or acetaldehyde coenzyme A dehydrogenase. Others, designated acd, were found to lack only acetaldehyde coenzyme A dehydrogenase. The acd mutation was located at min 62 of the E. coli genetic map, the gene order being thyA-lysA-acd-serA-fda. Isolation of Tn10 insertions cotransducible with acd greatly simplified the mapping procedure.