Halarcobacter arenosus sp. nov., isolated from marine sediment.

A Gram-stain-negative, slightly curved, rod-shaped bacterial strain CAU 1517T was isolated from marine sediment in Busan, the Republic of Korea. The taxonomic position of strain CAU 1517T was investigated via a polyphasic approach comprising phenotypic, chemotaxonomic and phylogenetic properties. Strain CAU 1517T grew optimally at 30 °C, pH 7.5 and in the presence of 7% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that strain CAU 1517T belongs to the genus Halarcobacter and is most closely related to Halarcobacter bivalviorum LMG 26154T (96.5% similarity). The average nucleotide identity and digital DNA-DNA hybridization values between strain CAU 1517T and members of genus Halarcobacter ranged from were 76.7-78.0% and 19.5-21.2%, respectively. The strain contained menaquinone-6 (MK-6) as the only respiratory quinone, and C16:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c), and summed feature 8 (C18:1ω7c/C18:1ω6c) as the major fatty acids. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylethanolamine, and two unidentified aminophospholipids. The G+C content was 28.2 mol%. Therefore, it has been demonstrated that the isolate represents a novel species of the genus Halarcobacter, for which the name Halarcobacter arenosus sp. nov., is proposed. The type strain is CAU 1517T (=KCTC 72232T =NBRC 113955T).

Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AIM: The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches. METHODS AND RESULTS: Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL-1 (10 fg). Direct DNA-based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family-level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL-1 . CONCLUSIONS: Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

Real-time quantitative PCR assay development and application for assessment of agricultural surface water and various fecal matter for prevalence of Aliarcobacter faecis and Aliarcobacter lanthieri.

BACKGROUND: Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL- 1 or g- 1 obtained using known quantities of spiked A. faecis and A. lanthieri reference strains in autoclaved agricultural surface water and dairy cow manure samples. RESULTS: Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of A. faecis for surface water and feces was 21 and 28%, respectively. The maximum A. faecis concentration for water and feces was 2.3 × 107 cells 100 mL- 1 and 1.2 × 107 cells g- 1, respectively. A. lanthieri was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 105 cells 100 mL- 1; fecal samples had a prevalence and maximum density of 10% and 2.0 × 106 cells g- 1, respectively. CONCLUSIONS: The results indicate that the occurrence of these species in agricultural surface water is potentially due to fecal contamination of water from livestock, human, or wildlife as both species were detected in fecal samples. The new real-time qPCR assays can facilitate rapid and accurate detection in < 3 h to quantify total numbers of A. faecis and A. lanthieri cells present in various complex environmental samples.

Wastewater treatment alters microbial colonization of microplastics.

Microplastics are ubiquitous contaminants in aquatic habitats globally, and wastewater treatment plants (WWTPs) are point sources of microplastics. Within aquatic habitats microplastics are colonized by microbial biofilms, which can include pathogenic taxa and taxa associated with plastic breakdown. Microplastics enter WWTPs in sewage and exit in sludge or effluent, but the role that WWTPs play in establishing or modifying microplastic bacterial assemblages is unknown. We analyzed microplastics and associated biofilms in raw sewage, effluent water, and sludge from two WWTPs. Both plants retained >99% of influent microplastics in sludge, and sludge microplastics showed higher bacterial species richness and higher abundance of taxa associated with bioflocculation (e.g. Xanthomonas) than influent microplastics, suggesting that colonization of microplastics within the WWTP may play a role in retention. Microplastics in WWTP effluent included significantly lower abundances of some potentially pathogenic bacterial taxa (e.g. Campylobacteraceae) compared to influent microplastics; however, other potentially pathogenic taxa (e.g. Acinetobacter) remained abundant on effluent microplastics, and several taxa linked to plastic breakdown (e.g. Klebsiella, Pseudomonas, and Sphingomonas) were significantly more abundant on effluent compared to influent microplastics. These results indicate that diverse bacterial assemblages colonize microplastics within sewage and that WWTPs can play a significant role in modifying the microplastic-associated assemblages, which may affect the fate of microplastics within the WWTPs and the environment.

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.

Structural and functional analysis of an l-serine O-phosphate decarboxylase involved in norcobamide biosynthesis.

Structural diversity of natural cobamides (Cbas, B12 vitamers) is limited to the nucleotide loop. The loop is connected to the cobalt-containing corrin ring via an (R)-1-aminopropan-2-ol O-2-phosphate (AP-P) linker moiety. AP-P is produced by the l-threonine O-3-phosphate (l-Thr-P) decarboxylase CobD. Here, the CobD homolog SMUL_1544 of the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans was characterized as a decarboxylase that produces ethanolamine O-phosphate (EA-P) from l-serine O-phosphate (l-Ser-P). EA-P is assumed to serve as precursor of the linker moiety of norcobamides that function as cofactors in the respiratory reductive dehalogenase. SMUL_1544 (SmCobD) is a pyridoxal-5'-phosphate (PLP)-containing enzyme. The structural analysis of the SmCobD apoprotein combined with the characterization of truncated mutant proteins uncovered a role of the SmCobD N-terminus in efficient l-Ser-P conversion.

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.

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.

Biosynthesis, Synthesis, and Activities of Barnesin A, a NRPS-PKS Hybrid Produced by an Anaerobic Epsilonproteobacterium.

Despite the wealth of physiological knowledge and plentiful genomes available, only few natural products of anaerobic bacteria have been identified until today and even less have been linked to their biosynthetic gene cluster. Here, we analyzed a unique NRPS-PKS hybrid gene cluster from an anaerobic Epsilonproteobacterium ( Sulfurospirillum barnesii). Phylogenetic analysis of key biosynthetic genes, gene expression studies, and comparative metabolomics resulted in the identification of the first anoxically biosynthesized NRPS-PKS hybrid metabolite: a lipo-dipeptide with a vinylogous side chain, called barnesin A. The absolute structure was verified by a modular total synthesis, and barnesin and derivatives were found to have antimicrobial activity, as well as selective and nanomolar inhibitory activity, against pharmacological important cysteine proteases, such as cathepsin B.

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.

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.

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.

Phylogenetic and Functional Prokaryotic Diversity in the Hoito-Gol Mesothermal Mineral Spring (Eastern Savan Mountains. Buryat Renuhlie).

High-throughput sequencing was used for comparative analysis of microbial communities of the water and mat from the Hoito-Gol mesothermal mineral sulfide spring (Eastern Sayan Mountains, Buryat Republic). Activity of microbial communities was determined. While both spring biotopes were dominated by members of three bacterial phyla, Proteobacteria, Bacteroidetes, and Firmicutes, they differed drastically in the composition of predominant phylotypes (at the genus level). In the water, the organisms wide spread in aquatic'environments were predominant, mostly aerobic chemoorganotrophs of the generaAcinetobacter, Pe- dobacter, and Flavobacterium. In the microbial mat,;the organisms actively involved in the sulfur cycle predominated, including sulfur-reducing bacteria Sulfurospirillum, sulfate-reducing deltaproteobacteria, sulfur- oxidizing chemoautotrophic bacteria, anoxygenic phototrophic bacteria of,the phyla Chloroflexi and Chloro- bi, as well as purple bacteria belonging to the Q-, P--, and y-Proteobacteria. Microbial mats of the spring exhibited higher phylogenetic diversity compared to high-temperature mats containing photosynthetic microorganisms.