Refined experimental annotation reveals conserved corrinoid autotrophy in chloroform-respiring Dehalobacter isolates

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.

Isolation and Characterization of Dehalobacter sp. Strain TeCB1 Including Identification of TcbA: A Novel Tetra- and Trichlorobenzene Reductive Dehalogenase

“…However, strain PER-K23 has a truncated cbiH gene that was hypothesized to render this strain incapable of cobalamin biosynthesis, and therefore reliant on exogenous cobalamin supply (Rupakula et al, 2014). This hypothesis was further validated when strains UNSWDHB and CF, possessing a full-length cbiH gene (locus tag DCF50_RS14480 and UNSWDHB_RS14695, respectively) were shown to grow without exogenous cobalamin supply (Wong et al, 2016; Wang et al, 2017). Despite TeCB1 being closely related to strains PER-K23 and E1 (also incapable of de novo cobalamin biosynthesis), it possesses a full-length cbiH gene (A7D23_RS 03205), which could confer it the capability of growth without exogenous addition of cobalamins, however, this hypothesis needs experimental confirmation.…”

“…To date all sequenced Dehalobacter genomes contain a full complement of cobalamin biosynthetic genes (Rupakula et al, 2014; Wong et al, 2016; Wang et al, 2017). However, strain PER-K23 has a truncated cbiH gene that was hypothesized to render this strain incapable of cobalamin biosynthesis, and therefore reliant on exogenous cobalamin supply (Rupakula et al, 2014).…”

Purinyl-cobamide is a native prosthetic group of reductive dehalogenases

“…2b). The corrinoids extracted from BN-PAGE gels were analyzed using an UltiMate 3000 UPLC system in tandem with a high-resolution Exactive Plus Orbitrap mass spectrometer (Thermo Fisher) and a UV-Vis detector set to 361 nm as described 48 . Corrinoids were separated on a Hypersil Gold C18 column (Thermo Fisher, 1.9 μm pore size, 2.1 mm inner diameter × 50 mm length) at a flow rate of 0.2 mL min −1 at 30 °C using 2.5 mM ammonium acetate in water as eluent A and 100% methanol as eluent B.…”

Guided cobamide biosynthesis for heterologous production of reductive dehalogenases

“…), Geobacter lovleyi (Yan et al ., ) and Dehalobacter sp. strains TCA1, CF, DCA and UNSWDHB (Sun et al ., ; Tang et al ., ; Wong et al ., ; Wang et al ., ). The organohalide‐respiring Dehalobacter restrictus (Rupakula et al ., ) and Dehalococcoides mccartyi (Löffler et al ., ) are Cba auxotrophs that strictly depend on the uptake of Cbas from the environment.…”

“…Cba production has been proven for S. multivorans Keller et al, 2014) and S. halorespirans , D. hafniense strains Y51, JH1, Viet1 and PCE1 (Reinhold et al, 2012;Yan et al 2018), Geobacter lovleyi (Yan et al, 2012) and Dehalobacter sp. strains TCA1, CF, DCA and UNSWDHB (Sun et al, 2002;Tang et al, 2016;Wong et al, 2016;Wang et al, 2017). The organohalide-respiring Dehalobacter restrictus (Rupakula et al, 2013) and Dehalococcoides mccartyi (L€ offler et al, 2013) are Cba auxotrophs that strictly depend on the uptake of Cbas from the environment.…”