Bacteria
of the genus Dehalogenimonas respire
with vicinally halogenated alkanes via dihaloelimination.
We aimed to describe involved proteins and their supermolecular organization.
Metagenomic sequencing of a Dehalogenimonas-containing culture resulted in a 1.65 Mbp draft genome of Dehalogenimonas alkenigignens strain BRE15M. It contained
31 full-length reductive dehalogenase homologous genes (rdhA), but only eight had cognate rdhB gene coding for
membrane-anchoring proteins. Shotgun proteomics of cells grown with
1,2-dichloropropane as an electron acceptor identified 1152 proteins
representing more than 60% of the total proteome. Ten RdhA proteins
were detected, including a DcpA ortholog, which was the strongest
expressed RdhA. Blue native gel electrophoresis
(BNE) demonstrating maximum activity was localized in a protein complex
of 146–242 kDa. Protein mass spectrometry revealed the presence
of DcpA, its membrane-anchoring protein DcpB, two hydrogen uptake
hydrogenase subunits (HupL and HupS), an iron–sulfur protein
(HupX), and subunits of a redox protein with a molybdopterin-binding
motif (OmeA and OmeB) in the complex. BNE after protein solubilization
with different detergent concentrations revealed no evidence for an
interaction between the putative respiratory electron input module
(HupLS) and the OmeA/OmeB/HupX module. All detected RdhAs comigrated
with the organohalide respiration complex. Based on genomic and proteomic
analysis, we propose quinone-independent respiration in Dehalogenimonas.
Dehalobacter (Firmicutes) encompass obligate organohalide‐respiring bacteria used for bioremediation of groundwater contaminated with halogenated organics. Various aspects of their biochemistry remain unknown, including the identities and interactions of respiratory proteins. Here, we sequenced the genome of Dehalobacter sp. strain 8M and analysed its protein expression. Strain 8M encodes 22 reductive dehalogenase homologous (RdhA) proteins. RdhA D8M_v2_40029 (TmrA) was among the two most abundant proteins during growth with trichloromethane and 1,1,2‐trichloroethane. To examine interactions of respiratory proteins, we used blue native gel electrophoresis together with dehalogenation activity tests and mass spectrometry. The highest activities were found in gel slices with the highest abundance of TmrA. Protein distributions across gel lanes provided biochemical evidence that the large and small subunits of the membrane‐bound [NiFe] uptake hydrogenase (HupL and HupS) interacted strongly and that HupL/S interacted weakly with RdhA. Moreover, the interaction of RdhB and membrane‐bound b‐type cytochrome HupC was detected. RdhC proteins, often encoded in rdh operons but without described function, migrated in a protein complex not associated with HupL/S or RdhA. This study provides the first biochemical evidence of respiratory protein interactions in Dehalobacter, discusses implications for the respiratory architecture and advances the molecular comprehension of this unique respiratory chain.
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