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Journal of Bacteriology, June 2009, p. 3842-3851, Vol. 191, No. 12
0021-9193/09/$08.00+0     doi:10.1128/JB.00230-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

In Vivo Analysis of Cobinamide Salvaging in Rhodobacter sphaeroides Strain 2.4.1 {triangledown} ,{dagger}

Michael J. Gray and Jorge C. Escalante-Semerena*

Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received 20 February 2009/ Accepted 7 April 2009

The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B12). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ+ strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed.

* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin, 6478 Microbial Sciences Building, 1550 Linden Drive, Madison, WI 53706. Phone: (608) 262-7379. Fax: (608) 265-7909. E-mail: escalante{at}bact.wisc.edu

{triangledown} Published ahead of print on 17 April 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, June 2009, p. 3842-3851, Vol. 191, No. 12
0021-9193/09/$08.00+0     doi:10.1128/JB.00230-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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