Characterization of (per)chlorate-reducing bacteria

Some bacteria can use (per )chlorate as terminal electron acceptor for growth. These bacteria convert perchlorate via chlorate and chlorite into chloride and molecular oxygen. Oxygen formation in microbial respiration is unique. In this study two chlorate-reducing strains belonging to the species Pseudomonas chloritidismutans and a (per )chlorate -reducing strain Dechloromonas hortensis were isolated. The characterization of the chlorate-reducing strain AW-1, which was isolated from a bioreactor treating a chlorate and bromate polluted waste stream, enabled the comparison of denitrifying bacteria with (per)chlorate-reducing bacteria. Despite a very close phylogenetic relationship between strain AW-1 and two Pseudomonas stutzeri strains, the former was not able to use nitrate as electron acceptor, and the latter two were not able to use chlorate. These results confirmed that separate pathways exist for chlorate and nitrate reduction. In accordance, chlorite dismutase activity was only present in strain AW-1 and it was therefore proposed to name strain AW-1, Pseudomonas chloritidismutans sp. nov . P. chloritidismutans was differentiated from other described (per )chlorate -reducing bacteria, by its inability to use perchlorate.To get more insight into the pathway of chlorate reduction, the chlorate reductase of P. chloritidismutans was purified and characterized. The oxygen sensitive chlorate reductase showed only reductase activity with chlorate and bromate which was different from the purified (per)chlorate reductase of the (per)chlorate-reducing strain GR-1, which in addition showed activity with perchlorate, nitrate and Mn(V). The chlorate reductase of Ideonella dechloratans also showed no reductase activity with perchlorate. The cytoplasmic localization of the chlorate reductase of P. chloritidismutans was different from the periplasmic orientation of the (per )chlorate reductase of strain GR-1 and of the chlorate reductase of I. dechloratans . The native enzyme had a molecular mass of 167 kDa and consisted of 3 subunits of 97, 38 and 34 kDa. The same stoichiometry (α 1 β 1 γ 1 ) was found for the chlorate reductase of I. dechloratans but this was different from the stoichiometry found for the (per )chlorate reductase of strain GR-1 (α 3 β 3 ). The b -subunit of the chlorate reductase of P. chloritidismutans revealed sequence similarity to b-subunits of various other reductases or dehydrogenases. The chlorate reductase from P. chloritidismutans was found to contain Mo and Fe, as was the case for the reductase of strain GR-1. EPR analysis confirmed this. The presence of molybdenum is not unusual because sofar, all enzymes that show chlorate reductase activity contain molybdenum (chlorate reductase of P. chloritidismutans and I. dechloratans , (per )chlorate reductase of strain GR-1, nitrate reductase, trimethylamine N-oxide (TMAO) reductase). These enzymes are positioned in the dimethyl sulfoxide (DMSO) reductase family. The chlorite dismutase was purified from P. chloritidismutans and this enzyme showed similar characteristics when compared to chlorite dismutases of strain GR-1, Dechloromonas agitata and I. dechloratans . The localization (periplasm), subunit composition (homotetramer), subunit size and the spectral characteristics (heme group, Fe-protoporphyrin IX) were the same. Experiments with 18 O-labelled water showed that no 36 O 2 or 34 O 2 labeled oxygen was produced, indicating that water is not a substrate in the conversion of chlorite and that oxygen is entirely produced out of chlorite. The N-terminal sequence of the chlorite dismutase of P. chloritidismutans had no similarity with the N-terminal sequences of other chlorite dismutases. Instead, similarity was found with an internal sequence of a hypothetical protein of dechloratans The gene coding for this hypothetical protein (partial ORF2) was located downstream of the gene of the chlorite dismutase. PCR experiments showed that the chlorite dismutase gene arrangement of P. chloritidismutans was different from the organization of the chlorite dismutase gene and partial ORF2 which was found for dechloratansThe previously isolated strain GR-1 had not yet been taxonomically described. Therefore, 16S rDNA sequence analysis and DNA-DNA hybridization experiments were performed. It was found that strain GR-1 was related at the species level to another (per )chlorate -reducing strain, Dechlorosoma suillum , which was recently renamed as Azospira oryzae . The occurrence of (per )chlorate -reducing bacteria in certain environments is a result of the contamination with (per)chlorate by anthropogenic activities. However, in this study also the possibility of (a )biotic processes by which chlorate can be formed is discussed.