Isolation and characterization of Dehalobacter sp. strain UNSWDHB capable of chloroform and chlorinated ethane respiration

Abstract

Dehalobacter sp. strain UNSWDHB can dechlorinate up to 4 mM trichloromethane at a rate of 0.1 mM per day to dichloromethane and 1,1,2-trichloroethane (1 mM, 0.1 mM per day) with the unprecedented product profile of 1,2-dichloroethane and vinyl chloride. 1,1,1-trichloroethane and 1,1-dichloroethane were slowly utilized by strain UNSWDHB and were not completely removed, with minimum threshold concentrations of 0.12 mM and 0.07 mM respectively under growth conditions. Enzyme kinetic experiments confirmed strong substrate affinity for trichloromethane and 1,1,2-trichloroethane (Km = 30 and 62 µM respectively) and poor substrate affinity for 1,1,1-trichloroethane and 1,1-dichloroethane (Km = 238 and 837 µM respectively). Comparison of enzyme kinetic and growth data with other trichloromethane respiring organisms (Dehalobacter sp. strain CF and Desulfitobacterium sp. strain PR) suggests an adaptation of strain UNSWDHB to trichloromethane. The trichloromethane RDase (TmrA) expressed by strain UNSWDHB was identified by BN-PAGE and functionally characterized. Amino acid comparison of homologous RDases from all three organisms revealed only six significant amino acid substitutions/deletions, which are likely to be crucial for substrate specificity. Furthermore, strain UNSWDHB was shown to grow without exogenous supply of cobalamin confirming genomic-based predictions of a fully functional cobalamin synthetic pathway.

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

“…Microscopic images of samples of both isolate cultures stained with 4′,6-diamidino-2-phenylindole dihydrochloride revealed a uniform cellular morphology, further supporting the culture purity (Figures 2a and b). Consistent with the three previously described Dehalobacter isolates (strains PER-K23, TCA1 and UNSWDHB; Holliger et al, 1998;Sun et al, 2002;Wong et al, 2016), the cells of strains CF and DCA are either straight or curved rods (Figures 2a and b) approximately 2-6 μm long with a diameter of 0.4-0.6 μm ( Figure 2c). Similar to strain PER-K23 (Holliger et al, 1998), strain CF stains Gram-negative (Supplementary Figure S4), whereas it is phylogenetically Gram-positive.…”

“…Moreover, the fact that strain DCA is able to respire 1,1,2-TCA, and that DcrA is the only RDase detected in 1,1-DCA grown cells (Tang and Edwards, 2013) suggest DcrA also dechlorinates 1,1,2-TCA. 1,1,2-TCA is therefore a common substrate for four similar RDases, including CfrA and DcrA described above, TmrA from strain UNSWDHB (Wong et al, 2016), and CtrA from Desulfitobacterium sp. strain PR (Zhao et al, 2015).…”

“…As we could amend with higher concentrations, 1,1,1-TCA was used as the electron acceptor for strain CF in experiments to verify genome annotations. The genomes of strains CF, DCA and UNSWDHB contain a complete operon (pceABCT) in their genomes encoding an ortholog of the characterized tetrachloroethene (PCE) RDase PceA from strain PER-K23 (Tang et al, 2016;Wong et al, 2016) (489% amino-acid identity). However, none of the strains was found to dechlorinate PCE.…”

“…Reductive dechlorination and growth of organohalide-respiring bacteria are usually enhanced with corrinoid supplementation (Yan et al, 2013). It is reported that strains TCA1 (Sun et al, 2002) and UNSWDHB (Wong et al, 2016) grow without corrinoid supplementation. Strain CF could also grow without corrinoid supplementation (Figure 1b).…”

Ecophysiology and environmental distribution of organohalide-respiring bacteria

“…genomes, it would be interesting to study their response to corrinoid starvation. This has, however, not been investigated to date since sufficient corrinoid is always supplied during cultivation [55,63,269,450].…”