Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two-component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies.

Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE-respiring representatives of the genus, uncovered the genetic inactivation of a two-component system (TCS) in the OHR gene region of the natural mutants. The assumption that the TCS gene products serve as a PCE sensor that initiates gene transcription was supported by the constitutive low-level expression of the TCS operon in fumarate-adapted cells of Sulfurospirillum multivorans. Via RNA sequencing, eight transcriptional units were identified in the OHR gene region, which includes the TCS operon, the PCE reductive dehalogenase operon, the gene cluster for norcobamide biosynthesis, and putative accessory genes with unknown functions. The OmpR-family response regulator (RR) encoded in the TCS operon was functionally characterized by promoter-binding assays. The RR bound a cis-regulatory element that contained a consensus sequence of a direct repeat (CTATW) separated by 17 bp. Its location either overlapping the -35 box or 50 bp further upstream indicated different regulatory mechanisms. Sequence variations in the regulator binding sites identified in the OHR gene region were in accordance with differences in the transcript levels of the respective gene clusters forming the PCE regulon. The results indicate the presence of a fine-tuned regulatory network controlling PCE metabolism in dehalogenating Sulfurospirillum species, a group of metabolically versatile organohalide-respiring bacteria.

Rapid pyritization in the presence of a sulfur/sulfate-reducing bacterial consortium.

Sedimentary pyrite (FeS2) is commonly thought to be a product of microbial sulfate reduction and hence may preserve biosignatures. However, proof that microorganisms are involved in pyrite formation is still lacking as only metastable iron sulfides are usually obtained in laboratory cultures. Here we show the rapid formation of large pyrite spherules through the sulfidation of Fe(III)-phosphate (FP) in the presence of a consortium of sulfur- and sulfate-reducing bacteria (SRB), Desulfovibrio and Sulfurospirillum, enriched from ferruginous and phosphate-rich Lake Pavin water. In biomineralization experiments inoculated with this consortium, pyrite formation occurred within only 3 weeks, likely enhanced by the local enrichment of polysulfides around SRB cells. During this same time frame, abiotic reaction of FP with sulfide led to the formation of vivianite (Fe3(PO4)2·8H2O) and mackinawite (FeS) only. Our results suggest that rates of pyritization vs. vivianite formation are regulated by SRB activity at the cellular scale, which enhances phosphate release into the aqueous phase by increased efficiency of iron sulfide precipitation, and thus that these microorganisms strongly influence biological productivity and Fe, S and P cycles in the environment.

Pharmaceuticals and Personal Care Products Can Be Transformed by Anaerobic Microbiomes in the Environment and in Waste-Treatment Processes.

Pharmaceuticals and personal care products (PPCPs) are emerging environmental contaminants that can be transformed by anaerobic microorganisms in anoxic environments. The present study examined 2 consortia, enriched under methanogenic and sulfate-rich conditions, that demethylate the phenylmethyl ether anti-inflammatory drug naproxen to 6-O-desmethylnaproxen. Both enriched consortia were also able to demethylate a range of phenylmethyl ether compounds of plant-based origin or used as PPCPs. Results from 16S rRNA gene sequencing showed that the 2 communities were very different despite sharing the same PPCP metabolism. In most cases, the demethylated metabolite was not further degraded but rather accumulated in the culture medium. For the expectorant guaifenesin, this resulted in a novel microbial metabolite. Furthermore, to our knowledge, this is the first report of methylparaben metabolism under methanogenic conditions. The wide range of phenylmethyl ether substrates that underwent O-demethylation in both methanogenic and sulfate-rich conditions suggests that there are potentially bioactive transformation products in the environment that have not yet been quantified. Environ Toxicol Chem 2019;38:1585-1593. © 2019 SETAC.

Hydrogen production by Sulfurospirillum species enables syntrophic interactions of Epsilonproteobacteria.

Hydrogen-producing bacteria are of environmental importance, since hydrogen is a major electron donor for prokaryotes in anoxic ecosystems. Epsilonproteobacteria are currently considered to be hydrogen-oxidizing bacteria exclusively. Here, we report hydrogen production upon pyruvate fermentation for free-living Epsilonproteobacteria, Sulfurospirillum spp. The amount of hydrogen produced is different in two subgroups of Sulfurospirillum spp., represented by S. cavolei and S. multivorans. The former produces more hydrogen and excretes acetate as sole organic acid, while the latter additionally produces lactate and succinate. Hydrogen production can be assigned by differential proteomics to a hydrogenase (similar to hydrogenase 4 from E. coli) that is more abundant during fermentation. A syntrophic interaction is established between Sulfurospirillum multivorans and Methanococcus voltae when cocultured with lactate as sole substrate, as the former cannot grow fermentatively on lactate alone and the latter relies on hydrogen for growth. This might hint to a yet unrecognized role of Epsilonproteobacteria as hydrogen producers in anoxic microbial communities.

A Retentive Memory of Tetrachloroethene Respiration in Sulfurospirillum halorespirans - involved Proteins and a possible link to Acetylation of a Two-Component Regulatory System.

Organohalide respiration (OHR), comprising the reductive dehalogenation of halogenated organic compounds, is subject to a unique memory effect and long-term transcriptional downregulation of the involved genes in Sulfurospirillum multivorans. Gene expression ceases slowly over approximately 100 generations in the absence of tetrachloroethene (PCE). However, the molecular mechanisms of this regulation process are not understood. We show here that Sulfurospirillum halorespirans undergoes the same type of regulation when cultivated without chlorinated ethenes for a long period of time. In addition, we compared the proteomes of S. halorespirans cells cultivated in the presence of PCE with those of cells long- and short-term cultivated with nitrate as the sole electron acceptor. Important OHR-related proteins previously unidentified in S. multivorans include a histidine kinase, a putative quinol dehydrogenase membrane protein, and a PCE-induced porin. Since for some regulatory proteins a posttranslational regulation of activity by lysine acetylations is known, we also analyzed the acetylome of S. halorespirans, revealing that 32% of the proteome was acetylated in at least one condition. The data indicate that the response regulator and the histidine kinase of a two-component system most probably involved in induction of PCE respiration are highly acetylated during short-term cultivation with nitrate in the absence of PCE. SIGNIFICANCE: The so far unique long-term downregulation of organohalide respiration is now identified in a second species suggesting a broader distribution of this regulatory phenomenon. An improved protein extraction method allowed the identification of proteins most probably involved in transcriptional regulation of OHR in Sulfurospirillum spp. Our data indicate that acetylations of regulatory proteins are involved in this extreme, sustained standby-mode of metabolic enzymes in the absence of a substrate. This first published acetylome of Epsilonproteobacteria might help to study other ecologically or medically important species of this clade.

Coexistence of two distinct Sulfurospirillum populations respiring tetrachloroethene-genomic and kinetic considerations.

Two anaerobic bacterial consortia, each harboring a distinct Sulfurospirillum population, were derived from a 10 year old consortium, SL2, previously characterized for the stepwise dechlorination of tetrachloroethene (PCE) to cis-dichloroethene (cis-DCE) via accumulation of trichloroethene (TCE). Population SL2-1 dechlorinated PCE to TCE exclusively, while SL2-2 produced cis-DCE from PCE without substantial TCE accumulation. The reasons explaining the long-term coexistence of the populations were investigated. Genome sequencing revealed a novel Sulfurospirillum species, designated 'Candidatus Sulfurospirillum diekertiae', whose genome differed significantly from other Sulfurospirillum spp. (78%-83% ANI). Genome-wise, SL2-1 and SL2-2 populations are almost identical, but differences in their tetrachloroethene reductive dehalogenase sequences explain the distinct dechlorination patterns. An extended series of batch cultures were performed at PCE concentrations of 2-200 µM. A model was developed to determine their dechlorination kinetic parameters. The affinity constant and maximal growth rate differ between the populations: the affinity is 6- to 8-fold higher and the growth rate 5-fold lower for SL2-1 than SL2-2. Mixed cultivation of the enriched populations at 6 and 30 µM PCE showed that a low PCE concentration could be the driving force for both functional diversity of reductive dehalogenases and niche specialization of organohalide-respiring bacteria with overlapping substrate ranges.

Reductive tetrachloroethene dehalogenation in the presence of oxygen by Sulfurospirillum multivorans: physiological studies and proteome analysis.

Reductive dehalogenation of organohalides is carried out by organohalide-respiring bacteria (OHRB) in anoxic environments. The tetrachloroethene (PCE)-respiring Epsilonproteobacterium Sulfurospirillum multivorans is one of few OHRB able to respire oxygen. Therefore, we investigated the organism's capacity to dehalogenate PCE in the presence of oxygen, which would broaden the applicability to use S. multivorans, unlike other commonly oxygen-sensitive OHRB, for bioremediation, e.g. at oxic/anoxic interphases. Additionally, this has an impact on our understanding of the global halogen cycle. Sulfurospirillum multivorans performs dehalogenation of PCE to cis-1,2-dichloroethene at oxygen concentrations below 0.19 mg/L. The redox potential of the medium electrochemically adjusted up to +400 mV had no influence on reductive dehalogenation by S. multivorans in our experiments, suggesting that higher levels of oxygen impair PCE dechlorination by inhibiting or inactivating involved enzymes. The PCE reductive dehalogenase remained active in cell extracts of S. multivorans exposed to 0.37 mg/L oxygen for more than 96 h. Analysis of the proteome revealed that superoxide reductase and cytochrome peroxidase amounts increased with 5% oxygen in the gas phase, while the response to atmospheric oxygen concentrations involved catalase and hydrogen peroxide reductase. Taken together, our results demonstrate that reductive dehalogenation by OHRB is not limited to anoxic conditions.

Metabolic Reconstruction and Modeling Microbial Electrosynthesis.

Microbial electrosynthesis is a renewable energy and chemical production platform that relies on microbial cells to capture electrons from a cathode and fix carbon. Yet despite the promise of this technology, the metabolic capacity of the microbes that inhabit the electrode surface and catalyze electron transfer in these systems remains largely unknown. We assembled thirteen draft genomes from a microbial electrosynthesis system producing primarily acetate from carbon dioxide, and their transcriptional activity was mapped to genomes from cells on the electrode surface and in the supernatant. This allowed us to create a metabolic model of the predominant community members belonging to Acetobacterium, Sulfurospirillum, and Desulfovibrio. According to the model, the Acetobacterium was the primary carbon fixer, and a keystone member of the community. Transcripts of soluble hydrogenases and ferredoxins from Acetobacterium and hydrogenases, formate dehydrogenase, and cytochromes of Desulfovibrio were found in high abundance near the electrode surface. Cytochrome c oxidases of facultative members of the community were highly expressed in the supernatant despite completely sealed reactors and constant flushing with anaerobic gases. These molecular discoveries and metabolic modeling now serve as a foundation for future examination and development of electrosynthetic microbial communities.

Cobamide-mediated enzymatic reductive dehalogenation via long-range electron transfer.

The capacity of metal-containing porphyrinoids to mediate reductive dehalogenation is implemented in cobamide-containing reductive dehalogenases (RDases), which serve as terminal reductases in organohalide-respiring microbes. RDases allow for the exploitation of halogenated compounds as electron acceptors. Their reaction mechanism is under debate. Here we report on substrate-enzyme interactions in a tetrachloroethene RDase (PceA) that also converts aryl halides. The shape of PceA's highly apolar active site directs binding of bromophenols at some distance from the cobalt and with the hydroxyl substituent towards the metal. A close cobalt-substrate interaction is not observed by electron paramagnetic resonance spectroscopy. Nonetheless, a halogen substituent para to the hydroxyl group is reductively eliminated and the path of the leaving halide is traced in the structure. Based on these findings, an enzymatic mechanism relying on a long-range electron transfer is concluded, which is without parallel in vitamin B12-dependent biochemistry and represents an effective mode of RDase catalysis.

Complex interactions between potentially pathogenic, opportunistic, and resident bacteria emerge during infection on a reef-building coral.

Increased bacterial diversity on diseased corals can obscure disease etiology and complicate our understanding of pathogenesis. To untangle microbes that may cause white band disease signs from microbes responding to disease, we inoculated healthy Acropora cervicornis corals with an infectious dose from visibly diseased corals. We sampled these dosed corals and healthy controls over time for sequencing of the bacterial 16S region. Endozoicomonas were associated with healthy fragments from 4/10 colonies, dominating microbiomes before dosing and decreasing over time only in corals that displayed disease signs, suggesting a role in disease resistance. We grouped disease-associated bacteria by when they increased in abundance (primary vs secondary) and whether they originated in the dose (colonizers) or the previously healthy corals (responders). We found that all primary responders increased in all dosed corals regardless of final disease state and are therefore unlikely to cause disease signs. In contrast, primary colonizers in the families Pasteurellaceae and Francisellaceae increased solely in dosed corals that ultimately displayed disease signs, and may be infectious foreign bacteria involved in the development of disease signs. Moving away from a static comparison of diseased and healthy bacterial communities, we provide a framework to identify key players in other coral diseases.