Stoichiometry of the gene products from the tetrachloroethene reductive dehalogenase operon pceABCT

ABSTRACT: Organohalide respiration (OHR) is a bacterial anaerobic process in which halogenated compounds, e.g. tetrachloroethene (PCE), are used as terminal electron acceptors. Our model organisms are Dehalobacter restrictus strain PER-K23, an obligate OHR bacterium, and Desulfitobacterium hafniense strain TCE1, a bacterium with a versatile energy metabolism. The key catalytic enzyme is the tetrachloroethene reductive dehalogenase (PceA) that is, at genomic level, encoded in a highly conserved gene cluster (pceABCT) in both D. restrictus and D. hafniense strain TCE1. To date, the functions of PceA and PceT, a dedicated molecular chaperone for the maturation of PceA, are well defined. However, the role of PceB and PceC are still not elucidated. Here, we present a multilevel study aiming at deciphering the stoichiometry of the pceABCT individual gene products. The investigation was assessed at RNA level by reverse transcription and (quantitative) polymerase chain reaction, while at protein level, proteomic analyses based on parallel reaction monitoring were performed to quantify the PceA, B, C and T proteins in cell-free extracts as well as in soluble and membrane fractions of both strains using heavy-labelled reference peptides. At RNA level, our results confirmed the co-transcription of all pce gene products, while the quantitative analysis revealed a relative stoichiometry of the gene transcripts of pceA, pceB, pceC and pceT at approximately 1.0:3.0:0.1:0.1 in D. restrictus. This trend was not observed in D. hafniense strain TCE1, where no substantial difference was measured for the four genes. At proteomic level, a 2:1 stoichiometry of PceA and PceB was observed in the membrane fraction, and a low abundance of PceC in comparison to the other two proteins. In the soluble fraction, a 1:1 stoichiometry of PceA and PceT was identified. In summary, we show that the pce gene cluster is transcribed as an operon with, however, a level of transcription that differs for individual genes, an observation that could be explained by post-transcriptional events, such as RNA processing and differential RNA stability. Results at protein level suggest that PceA and PceB form a membrane-bound PceA2B protein complex, which, in contrast to the proposed model, seems to be devoid of PceC.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Bacteria Dehalobacter Restrictus

DISEASE(S): Disease Free

SUBMITTER: Adrien Schmid  

LAB HEAD: Christof Holliger

PROVIDER: PXD030941 | Pride | 2022-04-06

REPOSITORIES: Pride