Thermotoga lettingae can salvage cobinamide to synthesize vitamin B12.

We recently reported that the Thermotogales acquired the ability to synthesize vitamin B-12 by acquisition of genes from two distantly related lineages, Archaea and Firmicutes (K. S. Swithers et al., Genome Biol. Evol. 4: 730-739, 2012). Ancestral state reconstruction suggested that the cobinamide salvage gene cluster was present in the Thermotogales' most recent common ancestor. We also predicted that Thermotoga lettingae could not synthesize B-12 de novo but could use the cobinamide salvage pathway to synthesize B-12. In this study, these hypotheses were tested, and we found that Tt. lettingae did not synthesize B-12 de novo but salvaged cobinamide. The growth rate of Tt. lettingae increased with the addition of B-12 or cobinamide to its medium. It synthesized B-12 when the medium was supplemented with cobinamide, and no B-12 was detected in cells grown on cobinamide-deficient medium. Upstream of the cobinamide salvage genes is a putative B-12 riboswitch. In other organisms, B-12 riboswitches allow for higher transcriptional activity in the absence of B-12. When Tt. lettingae was grown with no B-12, the salvage genes were upregulated compared to cells grown with B-12 or cobinamide. Another gene cluster with a putative B-12 riboswitch upstream is the btuFCD ABC transporter, and it showed a transcription pattern similar to that of the cobinamide salvage genes. The BtuF proteins from species that can and cannot salvage cobinamides were shown in vitro to bind both B-12 and cobinamide. These results suggest that Thermotogales species can use the BtuFCD transporter to import both B-12 and cobinamide, even if they cannot salvage cobinamide.