Comparative Analysis of Antimicrobial Antibodies between Mild and Severe COVID-19.

Patients with 2019 coronavirus disease (COVID-19) exhibit a broad spectrum of clinical presentations. A person's antimicrobial antibody profile, as partially shaped by past infection or vaccination, can reflect the immune system health that is critical to control and resolve the infection. We performed an explorative immunoproteomics study using microbial protein arrays displaying 318 full-length antigens from 77 viruses and 3 bacteria. We compared antimicrobial antibody profiles between 135 patients with mild COVID-19 disease and 215 patients with severe disease in 3 independent cohorts from Mexico and Italy. Severe disease patients were older with higher prevalence of comorbidities. We confirmed that severe disease patients elicited a stronger anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) response. We showed that antibodies against HCoV-229E and HcoV-NL63 but not against HcoV-HKU1 and HcoV-OC43 were also higher in those who had severe disease. We revealed that for a set of IgG and IgA antibodies targeting coronaviruses, herpesviruses, and other respiratory viruses, a subgroup of patients with the highest reactivity levels had a greater incidence of severe disease compared to those with mild disease across all three cohorts. On the contrary, fewer antibodies showed consistent greater prevalence in mild disease in all 3 cohorts. IMPORTANCE The clinical presentations of COVID-19 range from asymptomatic to critical illness that may lead to intensive care or even death. The health of the immune system, as partially shaped by past infections or vaccinations, is critical to control and resolve the infection. Using an innovative protein array platform, we surveyed antibodies against hundreds of full-length microbial antigens from 80 different viruses and bacteria in COVID-19 patients from different geographic regions with mild or severe disease. We not only confirmed the association of severe COVID-19 disease with higher reactivity of antibody responses to SARS-CoV-2 but also uncovered known and novel associations with antibody responses against herpesviruses and other respiratory viruses. Our study represents a significant step forward in understanding the factors contributing to COVID-19 disease severity. We also demonstrate the power of comprehensive antimicrobial antibody profiling in deciphering risk factors for severe COVID-19. We anticipate that our approach will have broad applications in infectious diseases.

Virome-wide detection of natural infection events and the associated antibody dynamics using longitudinal highly-multiplexed serology.

Current methods for detecting infections either require a sample collected from an actively infected site, are limited in the number of agents they can query, and/or yield no information on the immune response. Here we present an approach that uses temporally coordinated changes in highly-multiplexed antibody measurements from longitudinal blood samples to monitor infection events at sub-species resolution across the human virome. In a longitudinally-sampled cohort of South African adolescents representing >100 person-years, we identify >650 events across 48 virus species and observe strong epidemic effects, including high-incidence waves of Aichivirus A and the D68 subtype of Enterovirus D earlier than their widespread circulation was appreciated. In separate cohorts of adults who were sampled at higher frequency using self-collected dried blood spots, we show that such events temporally correlate with symptoms and transient inflammatory biomarker elevations, and observe the responding antibodies to persist for periods ranging from ≤1 week to >5 years. Our approach generates a rich view of viral/host dynamics, supporting novel studies in immunology and epidemiology.

PepSeq: a fully in vitro platform for highly multiplexed serology using customizable DNA-barcoded peptide libraries.

PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject's pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1-2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data.

Comparative Microbiomics Analysis of Antimicrobial Antibody Response between Patients with Lung Cancer and Control Subjects with Benign Pulmonary Nodules.

BACKGROUND: CT screening can detect lung cancer early but suffers a high false-positive rate. There is a need for molecular biomarkers that can distinguish malignant and benign indeterminate pulmonary nodules (IPN) detected by CT scan. METHODS: We profiled antibodies against 901 individual microbial antigens from 27 bacteria and 29 viruses in sera from 127 lung adenocarcinoma (ADC), 123 smoker controls (SMC), 170 benign nodule controls (BNC) individuals using protein microarrays to identify ADC and BNC specific antimicrobial antibodies. RESULTS: Analyzing fourth quartile ORs, we found more antibodies with higher prevalence in the three BNC subgroups than in ADC or SMC. We demonstrated that significantly more anti-Helicobacter pylori antibodies showed higher prevalence in ADC relative to SMC. We performed subgroup analysis and found that more antibodies with higher prevalence in light smokers (≤20 pack-years) compared with heavy smokers (>20 pack-years), in BNC with nodule size >1 cm than in those with ≤1 cm nodules, and in stage I ADC than in stage II and III ADC. We performed multivariate analysis and constructed antibody panels that can distinguish ADC versus SMC and ADC versus BNC with area under the ROC curve (AUC) of 0.88 and 0.80, respectively. CONCLUSIONS: Antimicrobial antibodies have the potential to reduce the false positive rate of CT screening and provide interesting insight in lung cancer development. IMPACT: Microbial infection plays an important role in lung cancer development and the formation of benign pulmonary nodules.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses.

The SARS-CoV-2 proteome shares regions of conservation with endemic human coronaviruses (CoVs), but it remains unknown to what extent these may be cross-recognized by the antibody response. Here, we study cross-reactivity using a highly multiplexed peptide assay (PepSeq) to generate an epitope-resolved view of IgG reactivity across all human CoVs in both COVID-19 convalescent and negative donors. PepSeq resolves epitopes across the SARS-CoV-2 Spike and Nucleocapsid proteins that are commonly targeted in convalescent donors, including several sites also recognized in some uninfected controls. By comparing patterns of homologous reactivity between CoVs and using targeted antibody-depletion experiments, we demonstrate that SARS-CoV-2 elicits antibodies that cross-recognize pandemic and endemic CoV antigens at two Spike S2 subunit epitopes. We further show that these cross-reactive antibodies preferentially bind endemic homologs. Our findings highlight sites at which the SARS-CoV-2 response appears to be shaped by previous CoV exposures and which have the potential to raise broadly neutralizing responses.

Tungstate Control of Microbial Sulfidogenesis and Souring of the Engineered Environment.

Sulfide accumulation in oil reservoir fluids (souring) from the activity of sulfate-reducing microorganisms (SRM) is of grave concern because of the associated health and facility failure risks. Here, we present an assessment of tungstate as a selective and potent inhibitor of SRM. Dose-response inhibitor experiments were conducted with a number of SRM isolates and enrichments at 30-80 °C and an increase in the effectiveness of tungstate treatment at higher temperatures was observed. To explore mixed inhibitor treatment modes, we tested synergy or antagonism between several inhibitors with tungstate, and found synergism between WO42- and NO2-, while additive effects were observed with ClO4- and NO3-. We also evaluated SRM inhibition by tungstate in advective upflow oil-sand-packed columns. Although 2 mM tungstate was initially sufficient to inhibit sulfidogenesis, subsequent temporal CaWO4 precipitation resulted in loss of the bioavailable inhibitor from solution and a concurrent increase in effluent sulfide. Mixing 4 mM sodium carbonate with the 2 mM tungstate was enough to promote tungstate solubility to reach inhibitory concentrations, without precipitation, and completely inhibit SRM activity. Overall, we demonstrate the effectiveness of tungstate as a potent SRM inhibitor, particularly at higher temperatures, and propose a novel carbonate-tungstate formulation for application to soured oil reservoirs.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with an endemic human CoV.

A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay ('PepSeq') to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.

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.

Resistance and Resilience of Sulfidogenic Communities in the Face of the Specific Inhibitor Perchlorate.

Hydrogen sulfide is a toxic and corrosive gas, produced by the activity of sulfate-reducing microorganisms (SRM). Owing to the environmental, economic and human-health consequences of sulfide, there is interest in developing specific inhibitors of SRM. Recent studies have identified perchlorate as a promising emerging inhibitor. The aim of this work is to quantitatively dissect the inhibitory dynamics of perchlorate. Sulfidogenic mixed continuous-flow systems were treated with perchlorate. SRM number, sulfide production and community structure were monitored pre-, during and post-treatment. The data generated was compared to a simple mathematical model, where SRM growth slows as a result of inhibition. The experimental data supports the interpretation that perchlorate largely acts to suppress SRM growth rates, rendering planktonic SRM increasingly susceptible to wash-out. Surface-attachment was identified as an important parameter preventing SRM wash-out and thus governing inhibitory dynamics. Our study confirmed the lesser depletion of surface-attached SRM as compared to planktonic SRM during perchlorate treatment. Indirect effects of perchlorate (bio-competitive exclusion of SRM by dissimilatory perchlorate-reducing bacteria, DPRB) were also assayed by amending reactors with DPRB. Indeed, low concentrations of perchlorate coupled with DRPB amendment can drive sulfide concentrations to zero. Further, inhibition in a complex community was compared to that in a pure culture, highlighting similarities and differences between the two scenarios. Finally, we quantified susceptibility to perchlorate across SRM in various culture conditions, showing that prediction of complex behavior in continuous systems from batch results is possible. This study thus provides an overview of the sensitivity of sulfidogenic communities to perchlorate, as well as mechanisms underlying these patterns.

Mitigating Sulfidogenesis With Simultaneous Perchlorate and Nitrate Treatments.

Sulfide biogenesis (souring) in oil reservoirs is an extensive and costly problem. Nitrate is currently used as a souring inhibitor but often requires high concentrations and yields inconsistent results. Recently, perchlorate has displayed promise as a more potent inhibitor in lab scale studies. However, combining the two treatments to determine synergy and effectiveness in a dynamic system has never been tested. Nitrate inhibits perchlorate consumption by perchlorate reducing bacteria, suggesting that the combined treatment may allow deeper penetration of the perchlorate into the reservoir matrix. Furthermore, the metabolic intermediates of perchlorate and nitrate reduction (nitrite and chlorite, respectively) are synergistic with the primary electron acceptors for inhibition of sulfate reduction. To assess the possible synergies between nitrate and perchlorate treatments, triplicate glass columns packed with pre-soured marine sediment were flushed with media containing sulfate and an inhibitor treatment [(i) perchlorate; (ii) nitrate; (iii) perchlorate and nitrate; or (iv) none]. Internal geochemistry and microbial community changes were monitored along the length of the columns during six phases of increasing treatment concentrations. In a final phase all treatments were removed. Sulfide production decreased in all treated columns in conjunction with increased inhibitor concentrations relative to the untreated control. Interestingly, the potency of the "mixed" treatment was additive relative to the individual treatments suggesting no interaction. Microbial community analyses indicated community shifts and clustering by treatment. The mixed treatment column community's trajectory closely resembled that of the community found in the perchlorate only treatment, suggesting that perchlorate was the dominant control on the "mixed" community structure. In contrast, the nitrate and untreated column communities had unique trajectories. This study indicates that concurrent nitrate and perchlorate treatment is not more effective than perchlorate treatment alone but is more effective than nitrate treatment. As such, treatment decisions may be based on economic factors.

Attenuating Sulfidogenesis in a Soured Continuous Flow Column System With Perchlorate Treatment.

Hydrogen sulfide production by sulfate reducing bacteria (SRB) is the primary cause of oil reservoir souring. Amending environments with chlorate or perchlorate [collectively denoted (per)chlorate] represents an emerging technology to prevent the onset of souring. Recent studies with perchlorate reducing bacteria (PRB) monocultures demonstrated that they have the innate capability to enzymatically oxidize sulfide, thus PRB may offer an effective means of reversing souring. (Per)chlorate may be effective by (i) direct toxicity to SRB; (ii) competitive exclusion of SRB by PRB; or (iii) reversal of souring through re-oxidation of sulfide by PRB. To determine if (per)chlorate could sweeten a soured column system and assign a quantitative value to each of the mechanisms we treated columns flooded with San Francisco bay water with temporally decreasing amounts (50, 25, and 12.5 mM) of (per)chlorate. Geochemistry and the microbial community structure were monitored and a reactive transport model was developed, Results were compared to columns treated with nitrate or untreated. Souring was reversed by all treatments at 50 mM but nitrate-treated columns began to re-sour when treatment concentrations decreased (25 mM). Re-souring was only observed in (per)chlorate-treated columns when concentrations were decreased to 12.5 mM and the extent of re-souring was less than the control columns. Microbial community analyses indicated treatment-specific community shifts. Nitrate treatment resulted in a distinct community enriched in genera known to perform sulfur cycling metabolisms and genera capable of nitrate reduction. (Per)chlorate treatment enriched for (per)chlorate reducing bacteria. (Per)chlorate treatments only enriched for sulfate reducing organisms when treatment levels were decreased. A reactive transport model of perchlorate treatment was developed and a baseline case simulation demonstrated that the model provided a good fit to the effluent geochemical data. Subsequent simulations teased out the relative role that each of the three perchlorate inhibition mechanisms played during different phases of the experiment. These results indicate that perchlorate addition is an effective strategy for both souring prevention and souring reversal. It provides insight into which organisms are involved, and illuminates the interactive effects of the inhibition mechanisms, further highlighting the versatility of perchlorate as a sweetening agent.

Genome-resolved metagenomics identifies genetic mobility, metabolic interactions, and unexpected diversity in perchlorate-reducing communities.

Dissimilatory perchlorate reduction is an anaerobic respiratory pathway that in communities might be influenced by metabolic interactions. Because the genes for perchlorate reduction are horizontally transferred, previous studies have been unable to identify uncultivated perchlorate-reducing populations. Here we recovered metagenome-assembled genomes from perchlorate-reducing sediment enrichments and employed a manual scaffolding approach to reconstruct gene clusters for perchlorate reduction found within mobile genetic elements. De novo assembly and binning of four enriched communities yielded 48 total draft genomes. In addition to canonical perchlorate reduction gene clusters and taxa, a new type of gene cluster with an alternative perchlorate reductase was identified. Phylogenetic analysis indicated past exchange between these gene clusters, and the presence of plasmids with either gene cluster shows that the potential for gene transfer via plasmid persisted throughout enrichment. However, a majority of genomes in each community lacked perchlorate reduction genes. Putative chlorate-reducing or sulfur-reducing populations were dominant in most communities, supporting the hypothesis that metabolic interactions might result from perchlorate reduction intermediates and byproducts. Other populations included a novel phylum-level lineage (Ca. Muirbacteria) and epibiotic prokaryotes with no known role in perchlorate reduction. These results reveal unexpected genetic diversity, suggest that perchlorate-reducing communities involve substantial metabolic interactions, and encourage expanded strategies to further understand the evolution and ecology of this metabolism.

Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway.

Dissimilatory phosphite oxidation (DPO), a microbial metabolism by which phosphite (HPO32-) is oxidized to phosphate (PO43-), is the most energetically favorable chemotrophic electron-donating process known. Only one DPO organism has been described to date, and little is known about the environmental relevance of this metabolism. In this study, we used 16S rRNA gene community analysis and genome-resolved metagenomics to characterize anaerobic wastewater treatment sludge enrichments performing DPO coupled to CO2 reduction. We identified an uncultivated DPO bacterium, Candidatus Phosphitivorax (Ca. P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within the Deltaproteobacteria, which has no known cultured isolates. Genes for phosphite oxidation and for CO2 reduction to formate were found in the genome of Ca. P. anaerolimi, but it appears to lack any of the known natural carbon fixation pathways. These observations led us to propose a metabolic model for autotrophic growth by Ca. P. anaerolimi whereby DPO drives CO2 reduction to formate, which is then assimilated into biomass via the reductive glycine pathway.

Characterization of an anaerobic marine microbial community exposed to combined fluxes of perchlorate and salinity.

The recent recognition of the environmental prevalence of perchlorate and its discovery on Mars, Earth's moon, and in meteorites, in addition to its novel application to controlling oil reservoir sulfidogenesis, has resulted in a renewed interest in this exotic ion and its associated microbiology. However, while plentiful data exists on freshwater perchlorate respiring organisms, information on their halophilic counterparts and microbial communities is scarce. Here, we investigated the temporal evolving structure of perchlorate respiring communities under a range of NaCl concentrations (1, 3, 5, 7, and 10 % wt/vol) using marine sediment amended with acetate and perchlorate. In general, perchlorate consumption rates were inversely proportional to NaCl concentration with the most rapid rate observed at 1 % NaCl. At 10 % NaCl, no perchlorate removal was observed. Transcriptional analysis of the 16S rRNA gene indicated that salinity impacted microbial community structure and the most active members were in families Rhodocyclaceae (1 and 3 % NaCl), Pseudomonadaceae (1 NaCl), Campylobacteraceae (1, 5, and 7 % NaCl), Sedimenticolaceae (3 % NaCl), Desulfuromonadaceae (5 and 7 % NaCl), Pelobacteraceae (5 % NaCl), Helicobacteraceae (5 and 7 % NaCl), and V1B07b93 (7 %). Novel isolates of genera Sedimenticola, Marinobacter, Denitromonas, Azoarcus, and Pseudomonas were obtained and their perchlorate respiring capacity confirmed. Although the obligate anaerobic, sulfur-reducing Desulfuromonadaceae species were dominant at 5 and 7 % NaCl, their enrichment may result from biological sulfur cycling, ensuing from the innate ability of DPRB to oxidize sulfide. Additionally, our results demonstrated enrichment of an archaeon of phylum Parvarchaeota at 5 % NaCl. To date, this phylum has only been described in metagenomic experiments of acid mine drainage and is unexpected in a marine community. These studies identify the intrinsic capacity of marine systems to respire perchlorate and significantly expand the known diversity of organisms capable of this novel metabolism.

Reactive Transport Model of Sulfur Cycling as Impacted by Perchlorate and Nitrate Treatments.

Microbial souring in oil reservoirs produces toxic, corrosive hydrogen sulfide through microbial sulfate reduction, often accompanying (sea)water flooding during secondary oil recovery. With data from column experiments as constraints, we developed the first reactive-transport model of a new candidate inhibitor, perchlorate, and compared it with the commonly used inhibitor, nitrate. Our model provided a good fit to the data, which suggest that perchlorate is more effective than nitrate on a per mole of inhibitor basis. Critically, we used our model to gain insight into the underlying competing mechanisms controlling the action of each inhibitor. This analysis suggested that competition by heterotrophic perchlorate reducers and direct inhibition by nitrite produced from heterotrophic nitrate reduction were the most important mechanisms for the perchlorate and nitrate treatments, respectively, in the modeled column experiments. This work demonstrates modeling to be a powerful tool for increasing and testing our understanding of reservoir-souring generation, prevention, and remediation processes, allowing us to incorporate insights derived from laboratory experiments into a framework that can potentially be used to assess risk and design optimal treatment schemes.

A molecular survey of Australian and North American termite genera indicates that vertical inheritance is the primary force shaping termite gut microbiomes.

BACKGROUND: Termites and their microbial gut symbionts are major recyclers of lignocellulosic biomass. This important symbiosis is obligate but relatively open and more complex in comparison to other well-known insect symbioses such as the strict vertical transmission of Buchnera in aphids. The relative roles of vertical inheritance and environmental factors such as diet in shaping the termite gut microbiome are not well understood. RESULTS: The gut microbiomes of 66 specimens representing seven higher and nine lower termite genera collected in Australia and North America were profiled by small subunit (SSU) rRNA amplicon pyrosequencing. These represent the first reported culture-independent gut microbiome data for three higher termite genera: Tenuirostritermes, Drepanotermes, and Gnathamitermes; and two lower termite genera: Marginitermes and Porotermes. Consistent with previous studies, bacteria comprise the largest fraction of termite gut symbionts, of which 11 phylotypes (6 Treponema, 1 Desulfarculus-like, 1 Desulfovibrio, 1 Anaerovorax-like, 1 Sporobacter-like, and 1 Pirellula-like) were widespread occurring in ≥50% of collected specimens. Archaea are generally considered to comprise only a minority of the termite gut microbiota (<3%); however, archaeal relative abundance was substantially higher and variable in a number of specimens including Macrognathotermes, Coptotermes, Schedorhinotermes, Porotermes, and Mastotermes (representing up to 54% of amplicon reads). A ciliate related to Clevelandella was detected in low abundance in Gnathamitermes indicating that protists were either reacquired after protists loss in higher termites or persisted in low numbers across this transition. Phylogenetic analyses of the bacterial communities indicate that vertical inheritance is the primary force shaping termite gut microbiota. The effect of diet is secondary and appears to influence the relative abundance, but not membership, of the gut communities. CONCLUSIONS: Vertical inheritance is the primary force shaping the termite gut microbiome indicating that species are successfully and faithfully passed from one generation to the next via trophallaxis or coprophagy. Changes in relative abundance can occur on shorter time scales and appear to be an adaptive mechanism for dietary fluctuations.

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.

Mechanisms of direct inhibition of the respiratory sulfate-reduction pathway by (per)chlorate and nitrate.

We investigated perchlorate (ClO(4)(-)) and chlorate (ClO(3)(-)) (collectively (per)chlorate) in comparison with nitrate as potential inhibitors of sulfide (H(2)S) production by mesophilic sulfate-reducing microorganisms (SRMs). We demonstrate the specificity and potency of (per)chlorate as direct SRM inhibitors in both pure cultures and undefined sulfidogenic communities. We demonstrate that (per)chlorate and nitrate are antagonistic inhibitors and resistance is cross-inducible implying that these compounds share at least one common mechanism of resistance. Using tagged-transposon pools we identified genes responsible for sensitivity and resistance in Desulfovibrio alaskensis G20. We found that mutants in Dde_2702 (Rex), a repressor of the central sulfate-reduction pathway were resistant to both (per)chlorate and nitrate. In general, Rex derepresses its regulon in response to increasing intracellular NADH:NAD(+) ratios. In cells in which respiratory sulfate reduction is inhibited, NADH:NAD(+) ratios should increase leading to derepression of the sulfate-reduction pathway. In support of this, in (per)chlorate or nitrate-stressed wild-type G20 we observed higher NADH:NAD(+) ratios, increased transcripts and increased peptide counts for genes in the core Rex regulon. We conclude that one mode of (per)chlorate and nitrate toxicity is as direct inhibitors of the central sulfate-reduction pathway. Our results demonstrate that (per)chlorate are more potent inhibitors than nitrate in both pure cultures and communities, implying that they represent an attractive alternative for controlling sulfidogenesis in industrial ecosystems. Of these, perchlorate offers better application logistics because of its inhibitory potency, solubility, relative chemical stability, low affinity for mineral cations and high mobility in environmental systems.

Inhibition of microbial sulfate reduction in a flow-through column system by (per)chlorate treatment.

Microbial sulfate reduction is a primary cause of oil reservoir souring. Here we show that amendment with chlorate or perchlorate [collectively (per)chlorate] potentially resolves this issue. Triplicate packed columns inoculated with marine sediment were flushed with coastal water amended with yeast extract and one of nitrate, chlorate, or perchlorate. Results showed that although sulfide production was dramatically reduced by all treatments, effluent sulfide was observed in the nitrate (10 mM) treatment after an initial inhibition period. In contrast, no effluent sulfide was observed with (per)chlorate (10 mM). Microbial community analyses indicated temporal community shifts and phylogenetic clustering by treatment. Nitrate addition stimulated Xanthomonadaceae and Rhizobiaceae growth, supporting their role in nitrate metabolism. (Per)chlorate showed distinct effects on microbial community structure compared with nitrate and resulted in a general suppression of the community relative to the untreated control combined with a significant decrease in sulfate reducing species abundance indicating specific toxicity. Furthermore, chlorate stimulated Pseudomonadaceae and Pseudoalteromonadaceae, members of which are known chlorate respirers, suggesting that chlorate may also control sulfidogenesis by biocompetitive exclusion of sulfate-reduction. Perchlorate addition stimulated Desulfobulbaceae and Desulfomonadaceae, which contain sulfide oxidizing and elemental sulfur-reducing species respectively, suggesting that effluent sulfide concentrations may be controlled through sulfur redox cycling in addition to toxicity and biocompetitive exclusion. Sulfur isotope analyses further support sulfur cycling in the columns, even when sulfide is not detected. This study indicates that (per)chlorate show great promise as inhibitors of sulfidogenesis in natural communities and provides insight into which organisms and respiratory processes are involved.