Corrinoid Auxotrophy in the Obligate Organohalide-Respiring Dehalobacter restrictus strain PER-K23

Introduction: Corrinoids are an essential cofactor of reductive dehalogenases, the key enzymes of organohalide respiration (OHR). Dehalobacter restrictus strain PER-K23 is an obligate OHR bacterium able to conserve energy with tetrachloroethene, but is unable to de novo synthesize corrinoids. In contrast, genome analysis of D. restrictus revealed the presence of the complete corrinoid biosynthesis pathway. Objectives: To understand the corrinoid metabolism of D. restrictus at the level of biosynthesis, regulation and transport and compare it to other emerging Dehalobacter genomes Methods: Genomes of Dehalobacter spp. was obtained from the JGI. Proteomics were applied on D. restrictus cells cultivated in media supplemented with corrinoids spanning a range from 0 to 250 µg/l. Gene expression analysed using targeted reverse transcription and quantitative PCR. Results and Discussion: A non-functional cbiH gene All the genes required for corrinoid biosynthesis & transport were identified in D. restrictus. However, there was a 101-bp deletion in the precorrin-3B C17-methyltransferase encoding cbiH. This mutation is not present in the genome of other Dehalobacter strains indicating that a non-functional cbiH could be the possible cause of corrinoid auxotrophy in D. restrictus. Active regulation of corrinoid metabolism The expression of corrinoid biosynthetic genes in D. restrictus was verified experimentally to be regulated by cobalamin riboswitches. Analysis of corrinoid-starved D. restrictus cells revealed an increased transcription of the genes encoded directly downstream of the riboswitches. After corrinoid addition all operons were repressed, with most pronounced reduction for two operons, termed operon 1 and 2, encoding enzymes involved in corrinoid transport and salvaging only found in the genome of D. restrictus (73- and 346-fold, respectively). Overproduction of corrinoid-salvaging proteins (CbiZ) and transporters Proteomics of corrinoid-starved cells revealed on average a 45-fold over-production of operon 2 thereby reinforcing the trend from transcriptomics. Taken together, these data suggest that D. restrictus has lost its ability of de novo corrinoid synthesis and instead evolved a strategy for augmented corrinoid uptake and modification to fulfill its corrinoid requirement.