Genetic and sequence analyses of a Pseudomonas denitrificans DNA fragment containing two cob genes

Abstract

A genetic analysis of a 12-kb DNA fragment containing Pseudomonas denitrificans cob genes was performed by transposon-mediated insertional mutagenesis. The nucleotide sequence and genetic analysis have shown that a 4.8-kb DNA subfragment carried two cob genes (cobS and cobT). Biochemical data concerning the complemented cobS and cobT mutants suggested that the cobS product was involved in cobalt insertionmediating reactions and that the cobT product was involved in the transformation of precorrin-3 into cobyrinic acid.Cobalamin biosynthesis is considered to require between 20 and 30 steps to transform uroporphyrinogen III into cobalamin. Most of the steps for the conversion of uroporphyrinogen III into cobyrinic acid remain to be established, but they include at least eight C methylations, elimination of C-20, NADPH-dependent reduction of the macrocycle, decarboxylation of the C-12 acetate, methyl migration from C-li to C-12, and cobalt insertion (for details, see references 23 and 33). Based on the study of Pseudomonas denitrificans, an up-to-date cobalamin biosynthesis scheme is presented in the accompanying paper (12).We have published data on the cloning of at least 14 cob genes (7), which are grouped into four clusters on the P. denitrificans chromosome. Genetic and biochemical analyses allowed the identification of 13 cob genes: cobA to cobE on a 5.4-kb fragment from complementation group C (11) and cobF to cobM on an 8.7-kb fragment from complementation group A (10). With the objective of searching for and identifying all the cob genes, a detailed genetic study of the complementation groups has been undertaken. In particular, our investigation focused on a large DNA fragment which was reported to contain two complementation classes (7). We present here the genetic analysis of a 12-kb P. denitrificans DNA fragment from complementation group B (7) and the nucleotide sequence of a 4.8-kb subfragment. Only two cob genes were found. The steps in the cobalamin pathway at which the encoded Cob proteins might act were investigated.

Complete DNA Sequence and Detailed Analysis of the Yersinia pestis KIM5 Plasmid Encoding Murine Toxin and Capsular Antigen

“…ORF15 is highly similar (28% identity over 42% of the target protein) to the Pseudomonas denitrificans cobT gene product, and ORF15a is similar (42% identity over 75% of the target protein) to the P. denitrificans CobS protein (16). Interestingly, cobT and cobS are linked to each other yet are not located with other cob gene clusters in P. denitrificans (16). Furthermore, the two genes are arranged in reverse order compared to their relative locations in P. denitrificans.…”

Sulphoacetaldehyde acetyltransferase yields acetyl phosphate: purification from Alcaligenes defragrans and gene clusters in taurine degradation

“…defragrans is on the complementary strand, and it is seen on the complementary strand of the β-proteobacterium B. fungorum and of the contaminative, putative β-proteobacterial DNA from the L. major sequencing project. These homologues of cobT are no more closely related to one another than to cobT ; cobST seem to be encoded separately from the main cob cluster(s) [42]. Genes with homologies to tauABC Ec , but poorly grouped in a dendrogram, are found near each xsc gene found in genome sequencing projects.…”

“…This pta gene overlapped (32 bp) the 3h-end of an ORF on the complementary strand. The polypeptide derived from 828 bp of the complementary strand shared 42 % identity with a CobT (accession number AAA25793.1) [42].…”

COBALAMIN (COENZYME B12): Synthesis and Biological Significance

“…Genomic libraries of P. denitrificans were screened for clones that complemented the various mutants. Analysis of the clones revealed four gene clusters (37,39,(47)(48)(49). The locations of these clusters on the P. denitrificans chromosome are unknown.…”