Chlorine redox chemistry is not rare in biology

Chlorine is abundant in cells and biomolecules, yet the biology of chlorine oxidation and reduction is poorly understood. Some bacteria encode the enzyme chlorite dismutase (Cld), which detoxifies chlorite (CIO2-) by converting it to chloride (Cl-) and molecular oxygen (O2). Cld is highly specific for chlorite and aside from low hydrogen peroxide activity has no known alternative substrate. Here, we reasoned that because chlorite is an intermediate oxidation state of chlorine, Cld can be used as a biomarker for oxidized chlorine species in microorganisms and microbial habitats. Cld was abundant in metagenomes from soils and freshwater to water treatment systems. About 5% of bacterial and archaeal genera contain an organism encoding Cld in its genome, and within some genera Cld is nearly conserved. Cld has been subjected to extensive horizontal gene transfer, suggesting selection by chlorite is episodic yet strong. Cld was also used as a biomarker to predict genes related to chlorine redox chemistry. Genes found to have a genetic association with Cld include known genes for responding to reactive chlorine species and uncharacterized genes for transporters, regulatory elements, and putative oxidoreductases that present targets for future research. Cld was repeatedly co-located in genomes with genes for enzymes that can inadvertently reduce perchlorate (CIO4-) or chlorate (CIO3-), confirming that in nature (per)chlorate reduction does not only occur in specialized anaerobic respiratory metabolisms. The presence of Cld in genomes of obligate aerobes without such enzymes suggested that chlorite, like hypochlorous acid (HOCl), might be formed by oxidative processes within natural habitats. In summary, the comparative genomics of Cld has provided an atlas for a deeper understanding of chlorine oxidation and reduction reactions that are an underrecognized feature of biology.

A Multiplex Immunosensor for Detecting Perchlorate-Reducing Bacteria for Environmental Monitoring and Planetary Exploration

Perchlorate anions are produced by chemical industries and are important contaminants in certain natural ecosystems. Perchlorate also occurs in some natural and uncontaminated environments such as the Atacama Desert, the high Arctic or the Antarctic Dry Valleys, and is especially abundant on the surface of Mars. As some bacterial strains are capable of using perchlorate as an electron acceptor under anaerobic conditions, their detection is relevant for environmental monitoring on Earth as well as for the search for life on Mars. We have developed an antibody microarray with 20 polyclonal antibodies to detect perchlorate-reducing bacteria (PRB) strains and two crucial and highly conserved enzymes involved in perchlorate respiration: perchlorate reductase and chlorite dismutase. We determined the cross-reactivity, the working concentration, and the limit of detection of each antibody individually and in a multiplex format by Fluorescent Sandwich Microarray Immunoassay. Although most of them exhibited relatively high sensitivity and specificity, we applied a deconvolution method based on graph theory to discriminate between specific signals and cross-reactions from related microorganisms. We validated the system by analyzing multiple bacterial isolates, crude extracts from contaminated reactors and salt-rich natural samples from the high Arctic. The PRB detecting chip (PRBCHIP) allowed us to detect and classify environmental isolates as well as to detect similar strains by using crude extracts obtained from 0.5 g even from soils with low organic-matter levels (<103 cells/g of soil). Our results demonstrated that PRBCHIP is a valuable tool for sensitive and reliable detection of perchlorate-reducing bacteria for research purposes, environmental monitoring and planetary exploration.

Unexpected photosensitivity of the well-characterized heme enzyme chlorite dismutase

Chlorite dismutase is a heme enzyme that catalyzes the conversion of the toxic compound ClO2− (chlorite) to innocuous Cl− and O2. The reaction is a very rare case of enzymatic O–O bond formation, which has sparked the interest to elucidate the reaction mechanism using pre-steady-state kinetics. During stopped-flow experiments, spectroscopic and structural changes of the enzyme were observed in the absence of a substrate in the time range from milliseconds to minutes. These effects are a consequence of illumination with UV–visible light during the stopped-flow experiment. The changes in the UV–visible spectrum in the initial 200 s of the reaction indicate a possible involvement of a ferric superoxide/ferrous oxo or ferric hydroxide intermediate during the photochemical inactivation. Observed EPR spectral changes after 30 min reaction time indicate the loss of the heme and release of iron during the process. During prolonged illumination, the oligomeric state of the enzyme changes from homo-pentameric to monomeric with subsequent protein precipitation. Understanding the effects of UV–visible light illumination induced changes of chlorite dismutase will help us to understand the nature and mechanism of photosensitivity of heme enzymes in general. Furthermore, previously reported stopped-flow data of chlorite dismutase and potentially other heme enzymes will need to be re-evaluated in the context of the photosensitivity. Graphic abstract Illumination of recombinantly expressed Azospira oryzae Chlorite dismutase (AoCld) with a high-intensity light source, common in stopped-flow equipment, results in disruption of the bond between FeIII and the axial histidine. This leads to the enzyme losing its heme cofactor and changing its oligomeric state as shown by spectroscopic changes and loss of activity.

Preferential Use of the Perchlorate over the Nitrate in the Respiratory Processes Mediated by the Bacterium Azospira sp. OGA 24

Here we report the results obtained for a strain isolated from a polluted site and classified as Azospira sp. OGA 24. The capability of OGA 24 to utilize perchlorate and nitrate and the regulation of pathways were investigated by growth kinetic studies and analysis of messenger RNA (mRNA) expression of the genes of perchlorate reductase alpha subunit (pcrA), chlorite dismutase (cld), and periplasmic nitrate reductase large subunit (napA). In aerobic conditions and in a minimal medium containing 10 mM acetate as carbon source, 5.6 ± 0.34 mmol L−1 perchlorate or 9.7 ± 0.22 mmol L−1 nitrate were efficiently reduced during the growth with 10 mM of either perchlorate or nitrate. In anaerobiosis, napA was completely inhibited in the presence of perchlorate as the only electron acceptor, pcrA was barely detectable in nitrate-reducing conditions. The cell growth kinetics were in accordance with expression data, indicating a separation of nitrate and perchlorate respiration pathways. In the presence of both compounds, anaerobic nitrate consumption was reduced to 50% (4.9 ± 0.4 vs. 9.8 ± 0.15 mmol L−1 without perchlorate), while that of perchlorate was not affected (7.2 ± 0.5 vs. 6.9 ± 0.6 mmol L−1 without nitrate). Expression analysis confirmed the negative effect of perchlorate on nitrate respiration. Based on sequence analysis of the considered genes and 16S ribosomal gene (rDNA), the taxonomic position of Azospira sp. OGA 24 in the perchlorate respiring bacteria (PRB) group was further defined by classifying it in the oryzae species. The respiratory characteristics of OGA 24 strain make it very attractive in terms of potential applications in the bioremediation of environments exposed to perchlorate salts.