Abstract Two novel chlorinated alkane-respiring Dehalobacter restrictus strains CF and DCA were isolated from the same enrichment culture, ACT-3, and characterized. The closed genomes of these highly similar sister strains were previously assembled from metagenomic sequence data and annotated. The isolation of the strains enabled experimental verification of predicted annotations, particularly focusing on irregularities or predicted gaps in central metabolic pathways and cofactor biosynthesis. Similar to D. restrictus strain PER-K23, strains CF and DCA require arginine, histidine and threonine for growth, although the corresponding biosynthesis pathways are predicted to be functional. Using strain CF to experimentally verify annotations, we determined that the predicted defective serine biosynthesis pathway can be rescued with a promiscuous serine hydroxymethyltransferase. Strain CF grew without added thiamine although the thiamine biosynthesis pathway is predicted to be absent; intracellular thiamine diphosphate, the cofactor of carboxylases in central metabolism, was not detected in cell extracts. Thus, strain CF may use amino acids to replenish central metabolites, portending entangled metabolite exchanges in ACT-3. Consistent with annotation, strain CF possesses a functional corrinoid biosynthesis pathway, demonstrated by increasing corrinoid content during growth and guided cobalamin biosynthesis in corrinoid-free medium. Chloroform toxicity to corrinoid-producing methanogens and acetogens may drive the conservation of corrinoid autotrophy in Dehalobacter strains. Heme detection in strain CF cell extracts suggests the ‘archaeal’ heme biosynthesis pathway also functions in anaerobic Firmicutes. This study reinforces the importance of incorporating enzyme promiscuity and cofactor availability in genome-scale functional predictions and identifies essential nutrient interdependencies in anaerobic dechlorinating microbial communities.