Anion transport as a target of adaption to perchlorate in sulfate-reducing communities.

Inhibitors can be used to control the functionality of microbial communities by targeting specific metabolisms. The targeted inhibition of dissimilatory sulfate reduction limits the generation of toxic and corrosive hydrogen sulfide across several industrial systems. Sulfate-reducing microorganisms (SRM) are specifically inhibited by sulfate analogs, such as perchlorate. Previously, we showed pure culture SRM adaptation to perchlorate stress through mutation of the sulfate adenylyltransferase, a central enzyme in the sulfate reduction pathway. Here, we explored adaptation to perchlorate across unconstrained SRM on a community scale. We followed natural and bio-augmented sulfidogenic communities through serial transfers in increasing concentrations of perchlorate. Our results demonstrated that perchlorate stress altered community structure by initially selecting for innately more resistant strains. Isolation, whole-genome sequencing, and molecular biology techniques allowed us to define subsequent genetic mechanisms of adaptation that arose across the dominant adapting SRM. Changes in the regulation of divalent anion:sodium symporter family transporters led to increased intracellular sulfate to perchlorate ratios, allowing SRM to escape the effects of competitive inhibition. Thus, in contrast to pure-culture results, SRM in communities cope with perchlorate stress via changes in anion transport and its regulation. This highlights the value of probing evolutionary questions in an ecological framework, bridging the gap between ecology, evolution, genomics, and physiology.

Functional Redundancy in Perchlorate and Nitrate Electron Transport Chains and Rewiring Respiratory Pathways to Alter Terminal Electron Acceptor Preference.

Most dissimilatory perchlorate reducing bacteria (DPRB) are also capable of respiratory nitrate reduction, and preferentially utilize nitrate over perchlorate as a terminal electron acceptor. The similar domain architectures and phylogenetic relatedness of the nitrate and perchlorate respiratory complexes suggests a common evolutionary history and a potential for functionally redundant electron carriers. In this study, we identify key genetic redundancies in the electron transfer pathways from the quinone pool(s) to the terminal nitrate and perchlorate reductases in Azospira suillum PS (hereafter referred to as PS). We show that the putative quinol dehydrogenases, (PcrQ and NapC) and the soluble cytochrome electron carriers (PcrO and NapO) are functionally redundant under anaerobic growth conditions. We demonstrate that, when grown diauxically with both nitrate and perchlorate, the endogenous expression of NapC and NapO during the nitrate reduction phase was sufficient to completely erase any growth defect in the perchlorate reduction phase caused by deletion of pcrQ and/or pcrO. We leveraged our understanding of these genetic redundancies to make PS mutants with altered electron acceptor preferences. Deletion of the periplasmic nitrate reductase catalytic subunit, napA, led to preferential utilization of perchlorate even in the presence of equimolar nitrate, and deletion of the electron carrier proteins napQ and napO, resulted in concurrent reduction of nitrate and perchlorate. Our results demonstrate that nitrate and perchlorate respiratory pathways in PS share key functionally redundant electron transfer proteins and that mutagenesis of these proteins can be utilized as a strategy to alter the preferential usage of nitrate over perchlorate.

Biotechnological Applications of Microbial (Per)chlorate Reduction.

While the microbial degradation of a chloroxyanion-based herbicide was first observed nearly ninety years ago, only recently have researchers elucidated the underlying mechanisms of perchlorate and chlorate [collectively, (per)chlorate] respiration. Although the obvious application of these metabolisms lies in the bioremediation and attenuation of (per)chlorate in contaminated environments, a diversity of alternative and innovative biotechnological applications has been proposed based on the unique metabolic abilities of dissimilatory (per)chlorate-reducing bacteria (DPRB). This is fueled in part by the unique ability of these organisms to generate molecular oxygen as a transient intermediate of the central pathway of (per)chlorate respiration. This ability, along with other novel aspects of the metabolism, have resulted in a wide and disparate range of potential biotechnological applications being proposed, including enzymatic perchlorate detection; gas gangrene therapy; enhanced xenobiotic bioremediation; oil reservoir bio-souring control; chemostat hygiene control; aeration enhancement in industrial bioreactors; and, biogenic oxygen production for planetary exploration. While previous reviews focus on the fundamental science of microbial (per)chlorate reduction (for example see Youngblut et al., 2016), here, we provide an overview of the emerging biotechnological applications of (per)chlorate respiration and the underlying organisms and enzymes to environmental and biotechnological industries.

Mechanism of H2S Oxidation by the Dissimilatory Perchlorate-Reducing Microorganism Azospira suillum PS.

The genetic and biochemical basis of perchlorate-dependent H2S oxidation (PSOX) was investigated in the dissimilatory perchlorate-reducing microorganism (DPRM) Azospira suillum PS (PS). Previously, it was shown that all known DPRMs innately oxidize H2S, producing elemental sulfur (So). Although the process involving PSOX is thermodynamically favorable (ΔG°' = -206 kJ â‹… mol-1 H2S), the underlying biochemical and genetic mechanisms are currently unknown. Interestingly, H2S is preferentially utilized over physiological electron donors such as lactate or acetate although no growth benefit is obtained from the metabolism. Here, we determined that PSOX is due to a combination of enzymatic and abiotic interactions involving reactive intermediates of perchlorate respiration. Using various approaches, including barcode analysis by sequencing (Bar-seq), transcriptome sequencing (RNA-seq), and proteomics, along with targeted mutagenesis and biochemical characterization, we identified all facets of PSOX in PS. In support of our proposed model, deletion of identified upregulated PS genes traditionally known to be involved in sulfur redox cycling (e.g., Sox, sulfide:quinone reductase [SQR]) showed no defect in PSOX activity. Proteomic analysis revealed differential abundances of a variety of stress response metal efflux pumps and divalent heavy-metal transporter proteins, suggesting a general toxicity response. Furthermore, in vitro biochemical studies demonstrated direct PSOX mediated by purified perchlorate reductase (PcrAB) in the absence of other electron transfer proteins. The results of these studies support a model in which H2S oxidation is mediated by electron transport chain short-circuiting in the periplasmic space where the PcrAB directly oxidizes H2S to So The biogenically formed reactive intermediates (ClO2- and O2) subsequently react with additional H2S, producing polysulfide and So as end products.IMPORTANCE Inorganic sulfur compounds are widespread in nature, and microorganisms are central to their transformation, thereby playing a key role in the global sulfur cycle. Sulfur oxidation is mediated by a broad phylogenetic diversity of microorganisms, including anoxygenic phototrophs and either aerobic or anaerobic chemotrophs coupled to oxygen or nitrate respiration, respectively. Recently, perchlorate-respiring microorganisms were demonstrated to be innately capable of sulfur oxidation regardless of their phylogenetic affiliation. As recognition of the prevalence of these organisms intensifies, their role in global geochemical cycles is being queried. This is further highlighted by the recently recognized environmental pervasiveness of perchlorate not only across Earth but also throughout our solar system. The inferred importance of this metabolism not only is that it is a novel and previously unrecognized component of the global sulfur redox cycle but also is because of the recently demonstrated applicability of perchlorate respiration in the control of biogenic sulfide production in engineered environments such as oil reservoirs and wastewater treatment facilities, where excess H2S represents a significant environmental, process, and health risk, with associated costs approximating $90 billion annually.

(Per)chlorate in Biology on Earth and Beyond.

Respiration of perchlorate and chlorate [collectively, (per)chlorate] was only recognized in the last 20 years, yet substantial advances have been made in our understanding of the underlying metabolisms. Although it was once considered solely anthropogenic, pervasive natural sources, both terrestrial and extraterrestrial, indicate an ancient (per)chlorate presence across our solar system. These discoveries stimulated interest in (per)chlorate microbiology, and the application of advanced approaches highlights exciting new facets. Forward and reverse genetics revealed new information regarding underlying molecular biology and associated regulatory mechanisms. Structural and functional analysis characterized core enzymes and identified novel reaction sequences. Comparative genomics elucidated evolutionary aspects, and stress analysis identified novel response mechanisms to reactive chlorine species. Finally, systems biology identified unique metabolic versatility and novel mechanisms of (per)chlorate respiration, including symbiosis and a hybrid enzymatic-abiotic metabolism. While many published studies focus on (per)chlorate and their basic metabolism, this review highlights seminal advances made over the last decade and identifies new directions and potential novel applications.

Phenotypic and genotypic description of Sedimenticola selenatireducens strain CUZ, a marine (per)chlorate-respiring gammaproteobacterium, and its close relative the chlorate-respiring Sedimenticola strain NSS.

Two (per)chlorate-reducing bacteria, strains CUZ and NSS, were isolated from marine sediments in Berkeley and San Diego, CA, respectively. Strain CUZ respired both perchlorate and chlorate [collectively designated (per)chlorate], while strain NSS respired only chlorate. Phylogenetic analysis classified both strains as close relatives of the gammaproteobacterium Sedimenticola selenatireducens. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations showed the presence of rod-shaped, motile cells containing one polar flagellum. Optimum growth for strain CUZ was observed at 25 to 30 °C, pH 7, and 4% NaCl, while strain NSS grew optimally at 37 to 42 °C, pH 7.5 to 8, and 1.5 to 2.5% NaCl. Both strains oxidized hydrogen, sulfide, various organic acids, and aromatics, such as benzoate and phenylacetate, as electron donors coupled to oxygen, nitrate, and (per)chlorate or chlorate as electron acceptors. The draft genome of strain CUZ carried the requisite (per)chlorate reduction island (PRI) for (per)chlorate respiration, while that of strain NSS carried the composite chlorate reduction transposon responsible for chlorate metabolism. The PRI of strain CUZ encoded a perchlorate reductase (Pcr), which reduced both perchlorate and chlorate, while the genome of strain NSS included a gene for a distinct chlorate reductase (Clr) that reduced only chlorate. When both (per)chlorate and nitrate were present, (per)chlorate was preferentially utilized if the inoculum was pregrown on (per)chlorate. Historically, (per)chlorate-reducing bacteria (PRB) and chlorate-reducing bacteria (CRB) have been isolated primarily from freshwater, mesophilic environments. This study describes the isolation and characterization of two highly related marine halophiles, one a PRB and the other a CRB, and thus broadens the known phylogenetic and physiological diversity of these unusual metabolisms.

Physiological and genetic description of dissimilatory perchlorate reduction by the novel marine bacterium Arcobacter sp. strain CAB.

A novel dissimilatory perchlorate-reducing bacterium (DPRB), Arcobacter sp. strain CAB, was isolated from a marina in Berkeley, CA. Phylogenetically, this halophile was most closely related to Arcobacter defluvii strain SW30-2 and Arcobacter ellisii. With acetate as the electron donor, strain CAB completely reduced perchlorate (ClO4(-)) or chlorate (ClO3(-)) [collectively designated (per)chlorate] to innocuous chloride (Cl(-)), likely using the perchlorate reductase (Pcr) and chlorite dismutase (Cld) enzymes. When grown with perchlorate, optimum growth was observed at 25 to 30°C, pH 7, and 3% NaCl. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations were dominated by free-swimming straight rods with 1 to 2 polar flagella per cell. Strain CAB utilized a variety of organic acids, fructose, and hydrogen as electron donors coupled to (per)chlorate reduction. Further, under anoxic growth conditions strain CAB utilized the biogenic oxygen produced as a result of chlorite dismutation to oxidize catechol via the meta-cleavage pathway of aerobic catechol degradation and the catechol 2,3-dioxygenase enzyme. In addition to (per)chlorate, oxygen and nitrate were alternatively used as electron acceptors. The 3.48-Mb draft genome encoded a distinct perchlorate reduction island (PRI) containing several transposases. The genome lacks the pcrC gene, which was previously thought to be essential for (per)chlorate reduction, and appears to use an unrelated Arcobacter c-type cytochrome to perform the same function. IMPORTANCE The study of dissimilatory perchlorate-reducing bacteria (DPRB) has largely focused on freshwater, mesophilic, neutral-pH environments. This study identifies a novel marine DPRB in the genus Arcobacter that represents the first description of a DPRB associated with the Campylobacteraceae. Strain CAB is currently the only epsilonproteobacterial DPRB in pure culture. The genome of strain CAB lacks the pcrC gene found in all other DPRB tested, demonstrating a new variation on the (per)chlorate reduction pathway. The ability of strain CAB to oxidize catechol via the oxygenase-dependent meta-cleavage pathway in the absence of external oxygen by using the biogenic oxygen produced from the dismutation of chlorite provides a valuable model for understanding the anaerobic degradation of a broad diversity of xenobiotics which are recalcitrant to anaerobic metabolism but labile to oxygenase-dependent mechanisms.