Tetrachloroethene Dehalogenase from Dehalospirillum multivorans: Cloning, Sequencing of the Encoding Genes, and Expression of the pceA Gene in Escherichia coli

ABSTRACT The genes encoding tetrachloroethene reductive dehalogenase, a corrinoid-Fe/S protein, of Dehalospirillum multivorans were cloned and sequenced. The pceA gene is upstream ofpceB and overlaps it by 4 bp. The presence of a ς70-like promoter sequence upstream of pceA and of a ρ-independent terminator downstream of pceB indicated that both genes are cotranscribed. This assumption is supported by reverse transcriptase PCR data. The pceA and pceB genes encode putative 501- and 74-amino-acid proteins, respectively, with calculated molecular masses of 55,887 and 8,354 Da, respectively. Four peptides obtained after trypsin treatment of tetrachloroethene (PCE) dehalogenase were found in the deduced amino acid sequence of pceA. The N-terminal amino acid sequence of the PCE dehalogenase isolated from D. multivorans was found 30 amino acids downstream of the N terminus of the deduced pceA product. The pceAgene contained a nucleotide stretch highly similar to binding motifs for two Fe4S4 clusters or for one Fe4S4 cluster and one Fe3S4 cluster. A consensus sequence for the binding of a corrinoid was not found in pceA. No significant similarities to genes in the databases were detected in sequence comparisons. The pceB gene contained two membrane-spanning helices as indicated by two hydrophobic stretches in the hydropathic plot. Sequence comparisons of pceBrevealed no sequence similarities to genes present in the databases. Only in the presence of pUBS 520 supplying the recombinant bacteria with high levels of the rare Escherichia colitRNA4 Arg was pceA expressed, albeit nonfunctionally, in recombinant E. coli BL21 (DE3).

Improved Dechlorinating Performance of Upflow Anaerobic Sludge Blanket Reactors by Incorporation ofDehalospirillum multivorans into Granular Sludge

ABSTRACT Dechlorination of tetrachloroethene, also known as perchloroethylene (PCE), was investigated in an upflow anaerobic sludge blanket (UASB) reactor after incorporation of the strictly anaerobic, reductively dechlorinating bacterium Dehalospirillum multivorans into granular sludge. This reactor was compared to the reference 1 (R1) reactor, where the granules were autoclaved to remove all dechlorinating abilities before inoculation, and to the reference 2 (R2) reactor, containing only living granular sludge. All three reactors were fed mineral medium containing 3 to 57 μM PCE, 2 mM formate, and 0.5 mM acetate and were operated under sterile conditions. In the test reactor, an average of 93% (mole/mole) of the effluent chloroethenes was dichloroethene (DCE), compared to 99% (mole/mole) in the R1 reactor. The R2 reactor, with no inoculation, produced only trichloroethene (TCE), averaging 43% (mole/mole) of the effluent chloroethenes. No dechlorination of PCE was observed in an abiotic control consisting of sterile granules without inoculum. During continuous operation with stepwise-reduced hydraulic retention times (HRTs), both the test reactor and the R1 reactor showed conversion of PCE to DCE, even at HRTs much lower than the reciprocal maximum specific growth rate of D. multivorans, indicating that this bacterium was immobilized in the living and autoclaved granular sludge. In contrast, the R2 reactor, with no inoculation of D. multivorans, only converted PCE to TCE under the same conditions. Immobilization could be confirmed by using fluorescein-labeled antibody probes raised against D. multivorans. In granules obtained from the R1 reactor, D. multivorans grew mainly in microcolonies located in the centers of the granules, while in the test reactor, the bacterium mainly covered the surfaces of granules.

Studies on the dechlorination of tetrachloroethene to cis-1,2-dichloroethene by dehalospirillum multivorans in biofilms

This study was conducted to examine the application of the anaerobic bacterium Dehalospirillum multivorans in a biofilm reactor for the reductive dechlorination of tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (DCE). A laboratory scale fluidized-bed reactor, operated under continuous flow conditions at 20°C, converted PCE to TCE at an apparent maximum reaction rate of 55 nmol/min/mg protein and TCE to DCE at 90 nmol/min/mg protein. These high rates should be even higher with an improved reactor performance since the apparent maximum reaction rates of the suspended cells (30°C) were by a factor of about 3.5 higher. With respect to its technical application D. multivorans is quite insensitive against unfavourable conditions, e.g. the presence of oxygen and low temperature. Natural groundwater components like nitrate and sulfate did not interfere with the dechlorination process up to a concentration of 5 mmol/l. Possible co-contaminants like chlorinated methanes, however, strongly inhibited PCE-dechlorination of the suspended cells, whereas chlorinated ethanes had no influence up to a concentration of 800 μmol/l. Experiments are presently being carried out to study the influence of biofilm immobilization on the protection of the dechlorination process against organic and inorganic inhibitors.