Stoichiometry of the Gene Products From the Tetrachloroethene Reductive Dehalogenase Operon pceABCT

Abstract

Organohalide respiration (OHR) is a bacterial anaerobic process that uses halogenated compounds, e.g., tetrachloroethene (PCE), as terminal electron acceptors. Our model organisms are Dehalobacter restrictus strain PER-K23, an obligate OHR bacterium (OHRB), and Desulfitobacterium hafniense strain TCE1, a bacterium with a versatile metabolism. The key enzyme is the PCE reductive dehalogenase (PceA) that is encoded in the highly conserved gene cluster (pceABCT) in both above-mentioned strains, and in other Firmicutes OHRB. 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. We present a multilevel study aiming at deciphering the stoichiometry of 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 Pce proteins in cell-free extracts as well as in soluble and membrane fractions of both strains using heavy-labeled reference peptides. At RNA level, our results confirmed the co-transcription of all pce genes, while the quantitative analysis revealed a relative stoichiometry of the gene transcripts of pceA, pceB, pceC, and pceT at ~ 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, an apparent 2:1 stoichiometry of PceA and PceB was obtained 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. Despite challenges in the quantification of integral membrane proteins such as PceB and PceC, the similar abundance of PceA and PceB invites to consider them as forming a membrane-bound PceA₂B protein complex, which, in contrast to the proposed model, seems to be devoid of PceC.

Keywords: Firmicutes (Bacillota); PRM quantitative proteomics; anaerobic respiration; gene product stoichiometry; operon; organohalogens; rdh gene clusters.

Figure 1

Co-transcription of pce genes in D. restrictus and D. hafniense strain TCE1. Individual genes and gene junctions were targeted by RT-PCR on RNA from (A) D. restrictus and (B) D. hafniense strain TCE1 cultivated with H₂ and PCE as electron donor and acceptor, respectively.

Figure 2

Quantitative analysis of the individual pce transcripts in (A) D. hafniense strain TCE1 cultivated on pyruvate and spiked with PCE; (B) D. hafniense strain TCE1 routinely cultivated with H₂ and PCE; (C) D. hafniense strain TCE1 routinely cultivated with pyruvate and PCE; and (D) D. restrictus routinely cultivated with H₂ and PCE. Please note that the transcriptional data of D. hafniense strain TCE1 were compared to transcripts obtained from cells routinely cultivated with pyruvate, while the gene transcription ratio of D. restrictus was calculated using DNA as reference.

Figure 3

Quantitative proteomics analysis of Pce proteins and the F1 α-subunit of the ATP synthase in cell-free extracts (CFE) from D. restrictus and D. hafniense strain TCE1. This graph shows the results of one biological replicate and is representative of the trend also observed for the second replicate (see Supplementary Table 2). The concentration of each protein was calculated by averaging the values obtained for the selected peptides in technical triplicates. Error bars indicate the calculated standard deviation.

Figure 4

Quantitative proteomics of the Pce proteins and the F1 α-subunit of the ATP synthase in sub-cellular compartments obtained from cell-free extracts of (A) D. restrictus and (B) D. hafniense strain TCE1. These graphs shows the results of one biological replicate and are representative of the trend also observed for the second replicate (see Supplementary Table 2). The concentration of each protein was calculated by averaging the values obtained for the selected peptides in technical triplicates. Error bars indicate the calculated standard deviation.