Iain C. Clark

Toward a mechanistic understanding of anaerobic nitrate-dependent iron oxidation: balancing electron uptake and detoxification

The anaerobic oxidation of Fe(II) by subsurface microorganisms is an important part of biogeochemical cycling in the environment, but the biochemical mechanisms used to couple iron oxidation to nitrate respiration are not well understood. Based on our own work and the evidence available in the literature, we propose a mechanistic model for anaerobic nitrate-dependent iron oxidation. We suggest that anaerobic iron-oxidizing microorganisms likely exist along a continuum including: (1) bacteria that inadvertently oxidize Fe(II) by abiotic or biotic reactions with enzymes or chemical intermediates in their metabolic pathways (e.g., denitrification) and suffer from toxicity or energetic penalty, (2) Fe(II) tolerant bacteria that gain little or no growth benefit from iron oxidation but can manage the toxic reactions, and (3) bacteria that efficiently accept electrons from Fe(II) to gain a growth advantage while preventing or mitigating the toxic reactions. Predictions of the proposed model are highlighted and experimental approaches are discussed.

Structure and Evolution of Chlorate Reduction Composite Transposons

ABSTRACT The genes for chlorate reduction in six bacterial strains were analyzed in order to gain insight into the metabolism. A newly isolated chlorate-reducing bacterium (Shewanella algae ACDC) and three previously isolated strains (Ideonella dechloratans, Pseudomonas sp. strain PK, and Dechloromarinus chlorophilus NSS) were genome sequenced and compared to published sequences (Alicycliphilus denitrificans BC plasmid pALIDE01 and Pseudomonas chloritidismutans AW-1). De novo assembly of genomes failed to join regions adjacent to genes involved in chlorate reduction, suggesting the presence of repeat regions. Using a bioinformatics approach and finishing PCRs to connect fragmented contigs, we discovered that chlorate reduction genes are flanked by insertion sequences, forming composite transposons in all four newly sequenced strains. These insertion sequences delineate regions with the potential to move horizontally and define a set of genes that may be important for chlorate reduction. In addition to core metabolic components, we have highlighted several such genes through comparative analysis and visualization. Phylogenetic analysis places chlorate reductase within a functionally diverse clade of type II dimethyl sulfoxide (DMSO) reductases, part of a larger family of enzymes with reactivity toward chlorate. Nucleotide-level forensics of regions surrounding chlorite dismutase (cld), as well as its phylogenetic clustering in a betaproteobacterial Cld clade, indicate that cld has been mobilized at least once from a perchlorate reducer to build chlorate respiration. IMPORTANCE Genome sequencing has identified, for the first time, chlorate reduction composite transposons. These transposons are constructed with flanking insertion sequences that differ in type and orientation between organisms, indicating that this mobile element has formed multiple times and is important for dissemination. Apart from core metabolic enzymes, very little is known about the genetic factors involved in chlorate reduction. Comparative analysis has identified several genes that may also be important, but the relative absence of accessory genes suggests that this mobile metabolism relies on host systems for electron transport, regulation, and cofactor synthesis. Phylogenetic analysis of Cld and ClrA provides support for the hypothesis that chlorate reduction was built multiple times from type II dimethyl sulfoxide (DMSO) reductases and cld. In at least one case, cld has been coopted from a perchlorate reduction island for this purpose. This work is a significant step toward understanding the genetics and evolution of chlorate reduction. Genome sequencing has identified, for the first time, chlorate reduction composite transposons. These transposons are constructed with flanking insertion sequences that differ in type and orientation between organisms, indicating that this mobile element has formed multiple times and is important for dissemination. Apart from core metabolic enzymes, very little is known about the genetic factors involved in chlorate reduction. Comparative analysis has identified several genes that may also be important, but the relative absence of accessory genes suggests that this mobile metabolism relies on host systems for electron transport, regulation, and cofactor synthesis. Phylogenetic analysis of Cld and ClrA provides support for the hypothesis that chlorate reduction was built multiple times from type II dimethyl sulfoxide (DMSO) reductases and cld. In at least one case, cld has been coopted from a perchlorate reduction island for this purpose. This work is a significant step toward understanding the genetics and evolution of chlorate reduction.

Identification of a perchlorate reduction genomic island with novel regulatory and metabolic genes.

ABSTRACT A comparative analysis of the genomes of four dissimilatory (per)chlorate-reducing bacteria has revealed a genomic island associated with perchlorate reduction. In addition to the characterized metabolic genes for perchlorate reductase and chlorite dismutase, the island contains multiple conserved uncharacterized genes possibly involved in electron transport and regulation.

Chlorate reduction in Shewanella algae ACDC is a recently acquired metabolism characterized by gene loss, suboptimal regulation and oxidative stress

Previous work on respiratory chlorate reduction has biochemically identified the terminal reductase ClrABC and the chlorite detoxifying enzyme Cld. In Shewanella algae ACDC, genes encoding these enzymes reside on composite transposons whose core we refer to as the chlorate reduction composite transposon interior (CRI). To better understand this metabolism in ACDC, we used RNA‐seq and proteomics to predict carbon and electron flow during chlorate reduction and posit that formate is an important electron carrier with lactate as the electron donor, but that NADH predominates on acetate. Chlorate‐specific transcription of electron transport chain components or the CRI was not observed, but clr and cld transcription was attenuated by oxygen. The major chlorate‐specific response related to oxidative stress and was indicative of reactive chlorine species production. A genetic system based on rpsL‐streptomycin counter selection was developed to further dissect the metabolism, but ACDC readily lost the CRI via homologous recombination of the composite transposons flanking insertion sequences. An engineered strain containing a single chromosomal CRI did not grow on chlorate, but overexpression of cld and its neighbouring cytochrome c restored growth. We postulate that the recently acquired CRI underwent copy‐number expansion to circumvent insufficient expression of key genes in the pathway.

Transposon and Deletion Mutagenesis of Genes Involved in Perchlorate Reduction in Azospira suillum PS

ABSTRACT Although much work on the biochemistry of the key enzymes of bacterial perchlorate reduction, chlorite dismutase, and perchlorate reductase has been published, understanding of the molecular mechanisms of this metabolism has been somewhat hampered by the lack of a clear model system amenable to genetic manipulation. Using transposon mutagenesis and clean deletions, genes important for perchlorate reduction in Azospira suillum PS have been identified both inside and outside the previously described perchlorate reduction genomic island (PRI). Transposon mutagenesis identified 18 insertions in 11 genes that completely abrogate growth via reduction of perchlorate but have no phenotype during denitrification. Of the mutants deficient in perchlorate reduction, 14 had insertions that were mapped to eight different genes within the PRI, highlighting its importance in this metabolism. To further explore the role of these genes, we also developed systems for constructing unmarked deletions and for complementing these deletions. Using these tools, every core gene in the PRI was systematically deleted; 8 of the 17 genes conserved in the PRI are essential for perchlorate respiration, including 3 genes that comprise a unique histidine kinase system. Interestingly, the other 9 genes in the PRI are not essential for perchlorate reduction and may thus have unknown functions during this metabolism. We present a model detailing our current understanding of perchlorate reduction that incorporates new concepts about this metabolism. IMPORTANCE Although perchlorate is generated naturally in the environment, groundwater contamination is largely a result of industrial activity. Bacteria capable of respiring perchlorate and remediating contaminated water have been isolated, but relatively little is known about the biochemistry and genetics of this process. Here we used two complementary approaches to identify genes involved in perchlorate reduction. Most of these genes are located on a genomic island, which is potentially capable of moving between organisms. Some of the genes identified are known to be directly involved in the metabolism of perchlorate, but other new genes likely regulate the metabolism in response to environmental signals. This work has uncovered new questions about the regulation, energetics, and evolution of perchlorate reduction but also presents the tools to address them. Although perchlorate is generated naturally in the environment, groundwater contamination is largely a result of industrial activity. Bacteria capable of respiring perchlorate and remediating contaminated water have been isolated, but relatively little is known about the biochemistry and genetics of this process. Here we used two complementary approaches to identify genes involved in perchlorate reduction. Most of these genes are located on a genomic island, which is potentially capable of moving between organisms. Some of the genes identified are known to be directly involved in the metabolism of perchlorate, but other new genes likely regulate the metabolism in response to environmental signals. This work has uncovered new questions about the regulation, energetics, and evolution of perchlorate reduction but also presents the tools to address them.

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

ABSTRACT 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.

Novel Mechanism for Scavenging of Hypochlorite Involving a Periplasmic Methionine-Rich Peptide and Methionine Sulfoxide Reductase

ABSTRACT Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and a putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo. IMPORTANCE Bacteria are often stressed in the environment by reactive chlorine species (RCS) of either anthropogenic or natural origin, but little is known of the defense mechanisms they have evolved. Using a microorganism that generates RCS internally as part of its respiratory process allowed us to uncover a novel defense mechanism based on RCS scavenging by reductive reaction with a sacrificial methionine-rich peptide and redox recycling through a methionine sulfoxide reductase. This system is conserved in a broad diversity of organisms, including some of clinical importance, invoking a possible important role in innate immune system evasion. Bacteria are often stressed in the environment by reactive chlorine species (RCS) of either anthropogenic or natural origin, but little is known of the defense mechanisms they have evolved. Using a microorganism that generates RCS internally as part of its respiratory process allowed us to uncover a novel defense mechanism based on RCS scavenging by reductive reaction with a sacrificial methionine-rich peptide and redox recycling through a methionine sulfoxide reductase. This system is conserved in a broad diversity of organisms, including some of clinical importance, invoking a possible important role in innate immune system evasion.

Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO32− bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

(Per)Chlorate-Reducing Bacteria Can Utilize Aerobic and Anaerobic Pathways of Aromatic Degradation with (Per)Chlorate as an Electron Acceptor

ABSTRACT The pathways involved in aromatic compound oxidation under perchlorate and chlorate [collectively known as (per)chlorate]-reducing conditions are poorly understood. Previous studies suggest that these are oxygenase-dependent pathways involving O2 biogenically produced during (per)chlorate respiration. Recently, we described Sedimenticola selenatireducens CUZ and Dechloromarinus chlorophilus NSS, which oxidized phenylacetate and benzoate, two key intermediates in aromatic compound catabolism, coupled to the reduction of perchlorate or chlorate, respectively, and nitrate. While strain CUZ also oxidized benzoate and phenylacetate with oxygen as an electron acceptor, strain NSS oxidized only the latter, even at a very low oxygen concentration (1%, vol/vol). Strains CUZ and NSS contain similar genes for both the anaerobic and aerobic-hybrid pathways of benzoate and phenylacetate degradation; however, the key genes (paaABCD) encoding the epoxidase of the aerobic-hybrid phenylacetate pathway were not found in either genome. By using transcriptomics and proteomics, as well as by monitoring metabolic intermediates, we investigated the utilization of the anaerobic and aerobic-hybrid pathways on different electron acceptors. For strain CUZ, the results indicated utilization of the anaerobic pathways with perchlorate and nitrate as electron acceptors and of the aerobic-hybrid pathways in the presence of oxygen. In contrast, proteomic results suggest that strain NSS may use a combination of the anaerobic and aerobic-hybrid pathways when growing on phenylacetate with chlorate. Though microbial (per)chlorate reduction produces molecular oxygen through the dismutation of chlorite (ClO2−), this study demonstrates that anaerobic pathways for the degradation of aromatics can still be utilized by these novel organisms. IMPORTANCE S. selenatireducens CUZ and D. chlorophilus NSS are (per)chlorate- and chlorate-reducing bacteria, respectively, whose genomes encode both anaerobic and aerobic-hybrid pathways for the degradation of phenylacetate and benzoate. Previous studies have shown that (per)chlorate-reducing bacteria and chlorate-reducing bacteria (CRB) can use aerobic pathways to oxidize aromatic compounds in otherwise anoxic environments by capturing the oxygen produced from chlorite dismutation. In contrast, we demonstrate that S. selenatireducens CUZ is the first perchlorate reducer known to utilize anaerobic aromatic degradation pathways with perchlorate as an electron acceptor and that it does so in preference over the aerobic-hybrid pathways, regardless of any oxygen produced from chlorite dismutation. D. chlorophilus NSS, on the other hand, may be carrying out anaerobic and aerobic-hybrid processes simultaneously. Concurrent use of anaerobic and aerobic pathways has not been previously reported for other CRB or any microorganisms that encode similar pathways of phenylacetate or benzoate degradation and may be advantageous in low-oxygen environments. S. selenatireducens CUZ and D. chlorophilus NSS are (per)chlorate- and chlorate-reducing bacteria, respectively, whose genomes encode both anaerobic and aerobic-hybrid pathways for the degradation of phenylacetate and benzoate. Previous studies have shown that (per)chlorate-reducing bacteria and chlorate-reducing bacteria (CRB) can use aerobic pathways to oxidize aromatic compounds in otherwise anoxic environments by capturing the oxygen produced from chlorite dismutation. In contrast, we demonstrate that S. selenatireducens CUZ is the first perchlorate reducer known to utilize anaerobic aromatic degradation pathways with perchlorate as an electron acceptor and that it does so in preference over the aerobic-hybrid pathways, regardless of any oxygen produced from chlorite dismutation. D. chlorophilus NSS, on the other hand, may be carrying out anaerobic and aerobic-hybrid processes simultaneously. Concurrent use of anaerobic and aerobic pathways has not been previously reported for other CRB or any microorganisms that encode similar pathways of phenylacetate or benzoate degradation and may be advantageous in low-oxygen environments.

Bioelectrical redox cycling of anthraquinone-2,6-disulfonate coupled to perchlorate reduction

Electrochemical reactors can be used to deliver electrons to bacteria with soluble, redox-active shuttles, and this has been proposed for both biosynthetic and bioremediation purposes. In the case of perchlorate reduction in bioelectrical reactors, very little is known about the mechanism of shuttle oxidation, although this understanding is important for treatment system design and scaleup. In this work, electrochemical, physiological, and proteomic experiments were performed to investigate anthrahydroquinone-2,6-disulfonate (AH2DS) oxidation by Azospira suillum PS under perchlorate reducing conditions. In inoculated bioelectrical reactors with AH2DS as the sole electron donor, only small amounts of cathodic current were observed. However, acetate addition resulted in a rapid increase in cathodic current, concomitant with acetate oxidation. This effect was not seen with nitrate as the terminal electron acceptor or when cells were lysed. We hypothesized that co-utilization of electron donors was a result of normal organotrophic perchlorate reduction, in addition to non-enzymatic reactions between AH2DS and intermediates (chlorite, oxygen) of the pathway. These reactions are expected to result in the production of reactive oxygen species (ROS) and toxicity, which was confirmed in batch growth experiments. Shotgun proteomics revealed significantly increased tandem spectral peptide counts of a diheme c-type cytochrome peroxidase; again, only when perchlorate, acetate, and AH2DS were present. Homologs of this protein relieve oxidative stress by reducing hydrogen peroxide to water. This work highlights the fact that oxidation of inorganic electron donors can occur non-enzymatically, as well as the challenge of targeting a specific metabolism with chemical shuttles that are highly reactive.