Proteome analysis of the response of Dehalobacter restrictus to cobalamin starvation

Dehalobacter restrictus strain PER-K23 is an obligate organohalide respiring microorganism known to solely use tetra- or trichloroethene as electron acceptors with H2 as electron donor. The catalytic enzyme, PceA, contains a corrinoid as cofactor, a trait believed to be general for reductive dehalogenases. Genomic studies showed the presence of both anaerobic cobalamin biosynthesis and cobinamide salvaging pathways, however, no growth was found without exogenous cobalamin supply. This observation is most likely due to a deletion in precorrin-3B C17-methyltransferase encoding gene cbiH (Rupakula et al., 2013, Philos Trans R Soc Lond B Biol Sci 368: 1616). In order to determine whether D. restrictus utilizes a salvaging or de novo synthesis strategy to obtain essential corrinoids under partial cobalamin starved conditions, D. restrictus was cultivated in the presence of either 250 (high), 50 (mid) or 10 (low) µg/l of cyanocobalamin. Presence and relative abundance of corrinoid-relevant proteins under the tested growth conditions was studied using shotgun proteomics. A total of 1195 proteins were detected from D. restrictus, corresponding to 42% of 2826 predicted protein coding genes in its genome. We detected CbiJ (precorrin-6x reductase, Dehre_0277), Cbi[ET] (precorrin-6Y methyltransferase, Dehre_2849) and CbiD (cobalamin biosynthesis protein, Dehre_2861) from upper cobalamin biosynthetic pathway, which were not detected in the previously analyzed proteome. However, several proteins belonging to the corrinoid synthesis pathway including CobS (cobalamin 5’-phosphate synthase, Dehre_1613), an alternative CbiJ (precorrin-6x reductase, Dehre_2855), CbiH (precorrin-3B C17-methyltransferase, Dehre_2856) and CbiG (cobalamin biosynthesis protein, Dehre_2858) were still missing from proteome. This suggests an incomplete cobalamin biosynthesis pathway that could explain why D. restrictus is incapable of de novo cobalamin biosynthesis. We identified proteins associated with corrinoid biosynthesis or salvaging pathways translated from five different operons. Proteins encoded by operon 2 showed the highest production levels with on average 45.7 fold up-regulation at low versus high cobalamin concentration, and included proteins predicted as members of corrinoid salvaging pathway and as corrinoid transporters. CbiZ proteins, amidohydrolase required for salvaging the cobalamin precursor cobinamide, showed up-regulation by 80 fold (Dehre_0285) and 58 fold (Dehre_0282) under cobalamin starvation, respectively. Furthermore, proteins encoded in operon 1 were on average 8.4 fold up-regulated between low and high cobalamin conditions, and included an ECF-type cobalt transporter (Dehre_0280), which is involved in the cobalt uptake process, and a precorrin-6x reductase (Dehre_0277). Genomic evidence suggests operon 1 and 2 being unique to D. restrictus in all so far available Dehalobacter strains, suggesting the response of operon 1 and 2 to changing cobalamin concentrations is exclusive to D. restrictus. In conclusion, proteomic data suggest that cobalamin biosynthesis in D. restrictus is non-functional and up-regulation of relevant cobalamin salvaging and transport pathways are the strategy employed to ensure sufficient amounts of corrinoids for growth.