Transcription and mass‐spectroscopic proteomic studies of electron transport oxidoreductases in Dehalococcoides ethenogenes

Abstract

Besides 19 potential reductive dehalogenase genes, the genome of Dehalococcoides ethenogenes strain 195 contains over 60 genes annotated as encoding oxidoreductases, including five hydrogenase complexes and a formate dehydrogenase (Fdh). Using quantitative reverse transcriptase polymerase chain reaction, we found that genes encoding a periplasmic Hup hydrogenase and the Fdh were the most highly expressed in batch-grown pure cultures, in which the H2 partial pressure was >0.1 atm, and in butyrate/tetrachloroethene-mixed cultures, in which H2 partial pressures were 10(-4)-10(-5) atm. Shotgun electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry were used to identify multiple peptides in pure culture membrane-enriched fractions matching several highly expressed respiratory enzymes, including three hydrogenases, two reductive dehalogenases, Fdh and DET1407, a 105.5-kDa protein we propose to be part of an S-layer cell wall. Both transcript and mass spectrometric approaches indicated that the putative Fdh was an important oxidoreductase in these cells; nevertheless, D. ethenogenes cultures could not use formate as an electron donor for reductive dechlorination. Analysis of the gene encoding the large subunit of Fdh indicated that while it was related to other Fdh proteins, its sequence encodes serine rather than cysteine or selenocysteine at a critical position, casting doubt on its function. Overall, genomic and proteomic approaches have provided novel insights into the metabolism of this difficult to culture organism.

The restricted metabolism of the obligate organohalide respiring bacterium Dehalobacter restrictus: lessons from tiered functional genomics

“…It was however observed that the expression of both Hyc and Ech decreased when Dehalococcoides mccartyi 195 was cultivated under lower partial pressure of hydrogen. Because hydrogen is a stronger reductant at higher partial pressure, the opposite would have been expected if they played a role in reverse electron flow [28]. Our findings suggest that different hydrogenases play specific and central roles in the metabolism of D. restrictus, but elucidating the exact role of the individual hydrogenases requires further studies.…”

“…This genus, although phylogenetically distant to Dehalobacter, inhabits similar ecological niches, and is exclusively dependent on OHR metabolism with H 2 as electron donor. These studies have used both transcriptomics using full genome microarrays and proteomics to identify key components of the metabolism of OHRB under different growth conditions or growth phases [23][24][25][26][27][28][29].…”

Global Transcriptomic and Proteomic Responses of Dehalococcoides ethenogenes Strain 195 to Fixed Nitrogen Limitation

“…Four of the hydrogenases (Hup, Ech, Hyc, and Hym) are membrane bound, while one (Vhu) is located in the cytoplasm (20,34,45). The Hup, Hym, and Vhu hydrogenase proteins were shown to be highly expressed in a previous study of a hydrogen-grown Dehalococcoides culture, suggesting that they are important for electron transport in dehalogenation, while the Ech and Hyc hy- drogenases are possibly important for developing low-potential electrons for biosynthetic reactions (34).…”

“…While bacteria from diverse genera can dechlorinate PCE and trichloroethene (TCE) to isomers of dichloroethene (DCE), to date, dechlorination beyond DCE to vinyl chloride (VC) and ethene has been observed only for strains of Dehalococcoides (47). Through transcriptional and protein analyses, the reductive dehalogenase (RDase)-and hydrogenase-encoding genes for energy generation in Dehalococcoides isolates and enrichments have been studied extensively (6,16,19,23,27,28,34,36,39,40,55). However, other aspects of the physiology of Dehalococcoides are not as well understood.…”

Investigation of Carbon Metabolism in “ Dehalococcoides ethenogenes ” Strain 195 by Use of Isotopomer and Transcriptomic Analyses

“…First, there are several key genes missing in the proposed amino acid biosynthetic pathways (e.g., methionine and glutamate) of the genome annotations (www.microbesonline.org), even though many Dehalococcoides isolates have been demonstrated to grow in minimal medium with only cysteine added as a reductant (1,7,8,33). Second, the current genome annotations predict that Dehalococcoides strains possess an incomplete Wood-Ljungdahl acetyl coenzyme A (acetyl-CoA) carbon fixation pathway that is missing a functional formate dehydrogenase, since the gene annotated with this function is apparently associated with a different function (22,23). Further, the subunit associated with carbon monoxide dehydrogenase activity in the carbon monoxide dehydrogenase/acetyl-CoA synthase/decarbonlyase (CODH/ACDS) enzyme complex is not annotated (31).…”