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. 1999 Nov;181(22):6907-13.

Anaerobic Growth of Paracoccus Denitrificans Requires Cobalamin: Characterization of cobK and cobJ Genes

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Free PMC article

Anaerobic Growth of Paracoccus Denitrificans Requires Cobalamin: Characterization of cobK and cobJ Genes

N Shearer et al. J Bacteriol. .
Free PMC article

Abstract

A pleiotropic mutant of Paracoccus denitrificans, which has a severe defect that affects its anaerobic growth when either nitrate, nitrite, or nitrous oxide is used as the terminal electron acceptor and which is also unable to use ethanolamine as a carbon and energy source for aerobic growth, was isolated. This phenotype of the mutant is expressed only during growth on minimal media and can be reversed by addition of cobalamin (vitamin B(12)) or cobinamide to the media or by growth on rich media. Sequence analysis revealed the mutation causing this phenotype to be in a gene homologous to cobK of Pseudomonas denitrificans, which encodes precorrin-6x reductase of the cobalamin biosynthesis pathway. Convergently transcribed with cobK is a gene homologous to cobJ of Pseudomonas denitrificans, which encodes precorrin-3b methyltransferase. The inability of the cobalamin auxotroph to grow aerobically on ethanolamine implies that wild-type P. denitrificans (which can grow on ethanolamine) expresses a cobalamin-dependent ethanolamine ammonia lyase and that this organism synthesizes cobalamin under both aerobic and anaerobic growth conditions. Comparison of the cobK and cobJ genes with their orthologues suggests that P. denitrificans uses the aerobic pathway for cobalamin synthesis. It is paradoxical that under anaerobic growth conditions, P. denitrificans appears to use the aerobic (oxygen-requiring) pathway for cobalamin synthesis. Anaerobic growth of the cobalamin auxotroph could be restored by the addition of deoxyribonucleosides to minimal media. These observations provide evidence that P. denitrificans expresses a cobalamin-dependent ribonucleotide reductase, which is essential for growth only under anaerobic conditions.

Figures

FIG. 1
FIG. 1
Map of a part of the cob locus of P. denitrificans. The approximate location of the original Tn5 insertion in the 9.6-kb PstI fragment that complements the Tn5 insertion mutant is indicated. The 2-kb PstI-SphI fragment that was sequenced is shown in expanded form, including the site of the Ω insertion (not to scale). Abbreviations: P, PstI; H, HindIII; B, BamHI; S, SphI. Nucleotide sequences from the regions where genes overlap are shown; start codons are indicated by arrows, stop codons are indicated by asterisks, and potential ribosome binding sites are underlined.
FIG. 2
FIG. 2
Alignment of CobK (precorrin-6x reductase) of P. denitrificans (Pde) with its orthologues from R. capsulatus (Rca [34]), Pseudomonas denitrificans (Psu [2]), M. tuberculosis (Mtu) (EMBL accession no. Q10680), and Rhodococcus sp. strain NI86/21 (Rho [8]). Included in the alignment are more distantly related precorrin-6x reductases from M. thermautotrophicum (Mth [30]), M. jannaschii (Mja [4]), B. megaterium (Bme [23]), S. typhimurium (Sty [24]), and Synechocystis sp. strain PCC6803 (Syn [16]), organisms which utilize the anaerobic pathway for cobalamin synthesis (23). Residues conserved only in enzymes from the aerobic pathway and only in enzymes from the anaerobic pathway are highlighted above (∗) and below (+) the alignment, respectively. Dashes indicate gaps introduced for alignment.
FIG. 3
FIG. 3
Alignment of CobJ (precorrin-3b methyltransferase) of P. denitrificans (Pde) with its orthologues from R. capsulatus (Rca [34]), Pseudomonas denitrificans (Psu [7]), and M. tuberculosis (Mtu) (SWISS-PROT accession no. Q10677). The M. tuberculosis sequence is the C-terminal portion of a fusion to precorrin-2 methyltransferase. Also in the alignment are more distantly related precorrin-3b methyltransferases from B. megaterium (Bme [23]), A. fulgidus (Afu [18]), M. jannaschii (Mja [4]), S. typhimurium (Sty [24]), and M. thermautotrophicum (Mth [30]). The A. fulgidus sequence is the N-terminal portion of a fusion to precorrin-8x methyltransferase. The M. thermautotrophicum protein has an additional 112 residues at its C terminus which are not shown. Residues conserved only in enzymes from the aerobic pathway and only in enzymes from the anaerobic pathway are highlighted above (∗) and below (+) the alignment, respectively. Regions corresponding to motifs I and III of SAM-dependent methyltransferases, as defined by Kagan and Clarke (15), are underlined. Dashes indicate gaps introduced for alignment.
FIG. 4
FIG. 4
Growth of Pd1222 and the cobalamin auxotroph NS52. Cultures of Pd1222 (open circles), NS52 (filled circles), and NS52 supplemented with cobalamin (squares) were grown anaerobically on succinate with either nitrate (50 mM), nitrite, or nitrous oxide as the terminal electron acceptor or aerobically with ethanolamine as the sole carbon and energy source. For growth on nitrite, the starting concentration was 3 mM and nitrite concentrations in the culture supernatants were monitored during growth. Periodic additions of nitrite were made to maintain the concentration at approximately 3 mM. For growth on nitrous oxide, bottles filled with media were sparged with N2O prior to inoculation and again at approximately 24-h intervals thereafter.

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