Ramlibacter terrae sp. nov. and Ramlibacter montanisoli sp. nov., Isolated from Soil.

Two gram-negative, catalase-positive, strictly aerobic, and white colony-forming bacteria, strains H242T and B156T, were isolated from soil in South Korea. Cells of strain H242T were oxidase-positive and non-motile short rods, while those of strain B156T were oxidase-negative and long non-motile rods. Ubiquinone-8 was identified as the sole isoprenoid quinone in both strains. C16:0, cyclo-C17:0, and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were identified in both strains as the major cellular fatty acids and polar lipids, respectively. The DNA G+C contents of strains H242T and B156T were 69.4 mol% and 69.3 mol%, respectively. Phylogenetic analyses based on 16S rRNA and 92 concatenated core gene sequences revealed that strains H242T and B156T formed distinct phylogenic lineages from other Ramlibacter type strains. The DNA-DNA hybridization (DDH) value between strains H242T and B156T was 24.6%. Strains H242T and B156T were most closely related to Ramlibacter ginsenosidimutans BXN5-27T and Ramlibacter monticola G-3-2T with 98.4% and 98.6% 16S rRNA gene sequence similarities, respectively. Digital DDH values between strain H242T and R. ginsenosidimutans and between strain B156T and R. monticola were 23.5% and 26.1%, respectively. Phenotypic, chemotaxonomic, and molecular analyses indicated that strains H242T and B156T represent two novel species of the genus Ramlibacter, for which the names Ramlibacter terrae sp. nov. and Ramlibacter montanisoli sp. nov., respectively, are proposed. The type strains of R. terrae and R. montanisoli are H242T (=KACC 21667 T =JCM 33922T) and B156T (=KACC 21665 T =JCM 33920T), respectively.

Molecular Basis for the Activation of Human Innate Immune Response by the Flagellin Derived from Plant-Pathogenic Bacterium, Acidovorax avenae.

Acidovorax avenae is a flagellated, pathogenic bacterium to various plant crops that has also been found in human patients with haematological malignancy, fever, and sepsis; however, the exact mechanism for infection in humans is not known. We hypothesized that the human innate immune system could be responsive to the purified flagellin isolated from A. avenae, named FLA-AA. We observed the secretion of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8 by treating FLA-AA to human dermal fibroblasts, as well as macrophages. This response was exclusively through TLR5, which was confirmed by using TLR5-overexpression cell line, 293/hTLR5, as well as TLR5-specific inhibitor, TH1020. We also observed the secretion of inflammatory cytokine, IL-1ß, by the activation of NLRC4 with FLA-AA. Overall, our results provide a molecular basis for the inflammatory response caused by FLA-AA in cell-based assays.

A Novel Antibacterial Component and the Mechanisms of an Amaranthus tricolor Leaf Ethyl Acetate Extract against Acidovorax avenae subsp. citrulli.

The aim of the present investigation was to determine the active ingredients in Amaranthus tricolor L. leaves and develop a biological pesticide. Organic solvent extraction, column chromatography, liquid chromatography, ODS-C18 reverse elution, Sephadex LH-20 gel filtration, H spectrum, and C spectrum were used to isolate the pure product for an assessment of the agricultural activity and bacteriostatic mechanisms. The results showed that the activity of the crude extract following carbon powder filtration was 1.63-fold that of the non-filtered extract. Further isolation was performed to obtain two pure products, namely, hydroxybenzoic acid (HBA) and benzo[b]furan-2-carboxaldehyde (BFC), and their molecular formulas and molecular weights were C7H6O3 and 138.12, and C9H6O2 and 146.12, respectively. Our study is the first to determine that HBA has bacteriostatic activity (MIC 125 µg/mL) and is also the first to isolate BFC from A. tricolor. The ultrastructure observation results showed that HBA caused the bacteria to become shriveled, distorted, and deformed, as well as exhibit uneven surfaces. After HBA treatment, 70 differentially expressed metabolites were detected in the bacteria, of which 9 were downregulated and 61 were upregulated. The differentially expressed metabolites were mainly strigolactones, organic acids and derivatives, fatty acids, benzene and substituted benzene derivatives, amino acids and associated metabolites, and alcohols and amines. Among all of the downregulated differentially expressed metabolites, MEDP1280 was the most critical, as it participates in many physiological and biochemical processes. The enrichment analysis showed that the differentially expressed metabolites mainly participate in tyrosine metabolism, biosynthesis of amino acids, cysteine and methionine metabolism, and arginine and proline metabolism. Additionally, HBA was found to disrupt cell membrane permeability and integrity, causing the leakage of substances and apoptosis. The physiological and biochemical test results showed that HBA could increase the pyruvate levels in bacteria but could decrease the activities of respiratory enzymes (malate dehydrogenase (MDH) and NADH oxidase) and antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX)). Inverse molecular docking was used to study the binding between HBA and respiratory and antioxidant enzymes. The results showed that HBA could bind to MDH, NADH oxidase, SOD, and GSH-PX, suggesting that these enzymes may be the effector targets of HBA. Conclusion: The optimal active ingredient in A. tricolor that can inhibit Acidovorax avenae subsp. citrulli was identified as HBA. HBA mainly disrupts the cell membrane, damages the metabolic system, and inhibits respiration and antioxidant enzyme activity to control bacterial growth. These results provide a reference for the further development of biological pesticides.

Reassembly of the Biosynthetic Gene Cluster Enables High Epothilone Yield in Engineered Schlegelella brevitalea.

Epothilones, as a new class of microtubule-stabilizing anticancer drugs, exhibit strong bioactivity against taxane-resistant cells and show clinical activity for the treatment of advanced breast cancer. Additionally, they also show great potential for a central nervous system injury and Alzheimer's disease. However, due to the long fermentation period of the original producer and challenges of genetic engineering of nonribosomal peptide/polyketide (NRP/PK) megasynthase genes, the application of epothilones is severely limited. Here, we addressed these problems by reassembling a novel 56-kb epothilone biosynthetic gene cluster, optimizing the promoter of each gene based on RNA-seq profiling, and completing precursor synthetic pathways in engineered Schlegella brevitalea. Furthermore, we debottlenecked the cell autolysis by optimizing culture conditions. Finally, the yield of epothilones in shake flasks was improved to 82 mg/L in six-day fermentation. Overall, we not only constructed epothilone overproducers for further drug development but also provided a rational strategy for high-level NRP/PK compound production.

Production of a novel dimeric 4-deoxy-L-erythro-5-hexoseulose uronic acid by a PL-17 exolytic alginate lyase from Hydrogenophaga sp. UMI-18.

Hydrogenopahaga sp. strain UMI-18 is an alginolytic bacterium that can produce poly(3-hydroxybutylate) (PHB) using alginate as its sole carbon source. Genome analysis indicated that this strain harbors both PHB-synthesizing and alginate-assimilating gene clusters. In the present study, we cloned HyAly-I gene that encodes a PL-17 exolytic alginate lyase and investigated its enzymatic properties using recombinant HyAly-I (recHyAly-I) that was produced by Escherichia coli. The recHyAly-I preferably depolymerized poly(ß-D-mannuronate) block of alginate in an exolytic manner at an optimal temperature and a pH at 40 °C and pH 6.0, respectively. It released 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from the non-reducing terminus of polymer and oligomer substrates. Interestingly, recHyAly-I was found to produce a novel unsaturated disaccharide, i.e., dimeric DEH (diDEH), along with monomeric DEH. Production of diDEH was prominent in the degradation of trisaccharides.

Extensimonas perlucida sp. nov., a Novel Bacterium Isolated from Sludge.

A bacterium, designated HX2-24 T, was isolated from activated sludge treating pesticide-manufacturing wastewater. Colonies of the strain on nutrient agar were circular, transparent, and colorless. Strain HX2-24 T shared 98.1% 16S rRNA gene sequence similarity with Extensimonas vulgaris S4T, and less than 97% similarities with other type strains. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain formed a clade with E. vulgaris S4T. The major cellular fatty acids were C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C17:0 cyclo, the major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), aminophospholipid (APL), glycophospholipid (GPL), and aminoglycolipid (AGL). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between HX2-24 T and E. vulgaris S4T were 92% and 41%, respectively. The G + C content of strain HX2-24 T was 64.4 mol%. Thus, based on the phenotypic, chemotaxonomic, and genotypic characteristics, strain HX2-24 T represents a novel species in the genus Extensimonas, for which the name Extensimonas perlucida HX2-24 T sp. nov. is proposed. The type strain is HX2-24 T (= KCTC 72472 T = CCTCC AB 2019178 T).

Proposal of three novel species of soil bacteria, Variovorax ureilyticus, Variovorax rhizosphaerae, and Variovorax robiniae, in the family Comamonadaceae.

Three novel bacterial strains (UCM-2T, UCM-G28T, and UCM-G35T) were obtained while isolating soil bacteria for the development of antibiotics. Cells of these strains were Gram-negative, non-spore forming, motile by means of a single flagellum, and rod shaped. In all strains, the predominant isoprenoid quinone was ubiquinone-8 (Q-8). Cells contained C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c), summed feature 8 (C18:1ω7c and/or C18:1ω6c), and C17:0 cyclo as the major fatty acids, and C10:0 3-OH as the major hydroxy fatty acid. The polar lipid profiles of the three novel strains were dominated by diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The genomic DNA G + C contents of strains UCM-2T, UCM-G28T, and UCMG35T were 67.5, 65.9, and 66.4 mol%, respectively. Phylogenetic analyses based on 16S rRNA sequences showed that strain UCM-2T was most closely related to Variovorax soli NBRC 106424T, whereas strains UCM-G28T and UCM-G35T were most similar to Variovorax ginsengisoli Gsoil 3165T. Values indicating DNA-DNA hybridization between the novel isolates and closely related species in the genus Variovorax were lower than the 70% cut-off point. These phenotypic, chemotaxonomic, and phylogenetic data indicate that the three isolates should be classified as new members of the genus Variovorax, for which the names Variovorax ureilyticus sp. nov., Variovorax rhizosphaerae sp. nov., and Variovorax robiniae sp. nov. are proposed. The type strains are UCM-2T (= KACC 18899T = NBRC 112306T), UCMG28T (= KACC 18900T = NBRC 112307T), and UCM-G35T (= KACC 18901T = NBRC 112308T), respectively.

Polyphasic characterization of four soil-derived phenanthrene-degrading Acidovorax strains and proposal of Acidovorax carolinensis sp. nov.

Four bacterial strains identified as members of the Acidovorax genus were isolated from two geographically distinct but similarly contaminated soils in North Carolina, USA, characterized, and their genomes sequenced. Their 16S rRNA genes were highly similar to those previously recovered during stable-isotope probing (SIP) of one of the soils with the polycyclic aromatic hydrocarbon (PAH) phenanthrene. Heterotrophic growth of all strains occurred with a number of organic acids, as well as phenanthrene, but no other tested PAHs. Optimal growth occurred aerobically under mesophilic temperature, neutral pH, and low salinity conditions. Predominant fatty acids were C16:1ω7c/C16:1ω6c, C16:0, and C18:1ω7c, and were consistent with the genus. Genomic G+C contents ranged from 63.6 to 64.2%. A combination of whole genome comparisons and physiological analyses indicated that these four strains likely represent a single species within the Acidovorax genus. Chromosomal genes for phenanthrene degradation to phthalate were nearly identical to highly conserved regions in phenanthrene-degrading Delftia, Burkholderia, Alcaligenes, and Massilia species in regions flanked by transposable or extrachromosomal elements. The lower degradation pathway for phenanthrene metabolism was inferred by comparisons to described genes and proteins. The novel species Acidovorax carolinensis sp. nov. is proposed, comprising the four strains described in this study with strain NA3T as the type strain (=LMG 30136, =DSM 105008).

Multidisciplinary characterization of U(VI) sequestration by Acidovorax facilis for bioremediation purposes.

The contamination of the environment by U may affect plant life and consequently may have an impact on animal and human health. The present work describes U(VI) sequestration by Acidovorax facilis using a multidisciplinary approach combining wet chemistry, transmission electron microscopy, and spectroscopy methods (e.g. cryo-time resolved laser-induced fluorescence spectroscopy, extended X-ray absorption fine structure spectroscopy, and in-situ attenuated total reflection Fourier transform infrared spectroscopy). This bacterial strain is widely distributed in nature including U-contaminated sites. In kinetic batch experiments cells of A. facilis were contacted for 5 min to 48 h with 0.1 mM U(VI). The results show that the local coordination of U species associated with the cells depends upon time contact. U is bound mainly to phosphate groups of lipopolysaccharide (LPS) at the outer membrane within the first hour. And, that both, phosphoryl and carboxyl functionality groups of LPS and peptidoglycan of A. facilis cells may effectuate the removal of high U amounts from solution at 24-48 h of incubation. It is clearly demonstrated that A. facilis may play an important role in predicting the transport behaviour of U in the environment and that the results will contribute to the improvement of bioremediation methods of U-contaminated sites.

Bifunctional quorum-quenching and antibiotic-acylase MacQ forms a 170-kDa capsule-shaped molecule containing spacer polypeptides.

Understanding the molecular mechanisms of bacterial antibiotic resistance will help prepare against further emergence of multi-drug resistant strains. MacQ is an enzyme responsible for the multi-drug resistance of Acidovorax sp. strain MR-S7. MacQ has acylase activity against both N-acylhomoserine lactones (AHLs), a class of signalling compounds involved in quorum sensing, and ß-lactam antibiotics. Thus, MacQ is crucial as a quencher of quorum sensing as well as in conferring antibiotic resistance in Acidovorax. Here, we report the X-ray structures of MacQ in ligand-free and reaction product complexes. MacQ forms a 170-kDa capsule-shaped molecule via face-to-face interaction with two heterodimers consisting of an α-chain and a ß-chain, generated by the self-cleaving activity of a precursor polypeptide. The electron density of the spacer polypeptide in the hollow of the molecule revealed the close orientation of the peptide-bond atoms of Val20SP-Gly21SP to the active-site, implying a role of the residues in substrate binding. In mutational analyses, uncleaved MacQ retained degradation activity against both AHLs and penicillin G. These results provide novel insights into the mechanism of self-cleaving maturation and enzymatic function of N-terminal nucleophile hydrolases.

Phosphate-binding protein from Polaromonas JS666: purification, characterization, crystallization and sulfur SAD phasing.

Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP from Polaromonas JS666 is focused on. Interestingly, this PBP is predicted to harbor different phosphate-binding residues to currently known PBPs. Here, it is shown that the PBP from Polaromonas JS666 is capable of binding phosphate, with a maximal binding activity at pH 8. Its structure is expected to reveal its binding-cleft configuration as well as its phosphate-binding mode. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.35 Šresolution of the PBP from Polaromonas JS666 are reported.

Acidovorax lacteus sp. nov., isolated from a culture of a bloom-forming cyanobacterium (Microcystis sp.).

A novel Gram-negative, rod-shaped and motile bacterial strain, designated strain M36T, was isolated from a culture of a bloom-forming cyanobacterium, Microcystis sp., collected from a eutrophic lake in Korea. Its taxonomic position was investigated by using a polyphasic taxonomic approach. The isolate was found to grow aerobically at 15-42 °C (optimum 25 °C), pH 7.0-11.0 (optimum pH 8.0) and in the presence of 0-1.0% (w/v) NaCl (optimum 0% NaCl) on R2A medium. The phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain M36T is closely related to Acidovorax anthurii DSM 16745T (98.1%), Acidovorax konjaci DSM 7481T (97.7%) and Acidovorax avenae DSM 7227T (97.0%) and also formed a clear phylogenetic lineage with other Acidovorax species. DNA-DNA relatedness between strain M36T and the closely related species of the genus Acidovorax was <30%. The major fatty acid components identified included summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0 and summed feature 8 (C18:0 ω7c and/or C18:0 ω6c). The DNA G+C content of strain M36T was determined to be 66.8 mol%. Based on above polyphasic evidence, strain M36T is concluded to represent a new species of genus Acidovorax, for which the name Acidovorax lacteus sp. nov. is proposed. The type strain is M36T (=KCTC 52220T = JCM 31890T).

Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel.

Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.

Kinneretia THG-SQI4 mediated biosynthesis of silver nanoparticles and its antimicrobial efficacy.

Simple, facile, effective approach for the biosynthesis of silver nanoparticles using Kinneretia species and its antimicrobial activity against human pathogens has been demonstrated in this study. Kinneretia THG-SQI4 has been isolated from soil sample collected from Shangqui, China. The synthesized nanoparticles were characterized by ultraviolet-visible spectrophotometry (UV-vis), field emission transmission electron microscopy (FE-TEM), energy dispersive X-ray spectroscopy (EDX), elemental mapping, selected area diffraction pattern, and X-ray diffraction (XRD). The synthesized silver nanoparticles were characterized by a peak at 425 nm in the UV-vis spectrum. The TEM data reveals that the nanoparticles are monodisperse and spherical in shape having a diameter ranging from 15 to 20 nm. The bright circular spots in SAED pattern confirm the crystalline nature of the nanoparticles. The XRD spectra exhibited the characteristic Bragg peaks of 1 1 1, 2 0 0, 2 2 0, and 3 1 1 facets of the face centered cubic symmetry of nanoparticles. The synthesized silver nanoparticles showed potent antibacterial activity against human pathogens like Candida albicans, Candida tropicalis, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Salmonella enterica, Pseudomonas aeruginosa, Escherichia coli, and Vibrio parahaemolyticus. Moreover, the silver nanoparticles show enhanced synergistic effects in combination with different standard antibiotics against Gram-negative bacteria. This method for synthesis of silver nanoparticle is valuable and has antimicrobial potential.

[Curvibacter sp. strain HJ-1 induced the formation of aragonite under the condition of low Mg/Ca ratio].

Objective: To study the effects of bacteria on the species and morphology of carbonate minerals. Methods: We conducted a series of cultural experiments in the medium with initial Mg/Ca ratio of 2 but without carbonate ion using Curvibacter sp. strain HJ-1 for 50 days. During the cultivation, bacterial density, precipitate quantities, calcium and magnesium concentration were determined. The morphologies of precipitated carbonates were observed using scanning electron microscopy, and mineral species of carbonate were determined by X-ray diffraction. Results: Strain HJ-1 could induce the precipitation of carbonate minerals, the quality of carbonate gradually increased with the incubation time. XRD patterns showed that the mineral precipitates consisted of high-Mg calcite and aragonite. The percentage of aragonite in the precipitates was up to 86%. The morphology of carbonate minerals was multiform, including rod-shaped, dumbbell-shaped, spherical, tabular, as well as irregular and flake. Conclusion: The formation of aragonite under the condition of low Mg/Ca ratio has a close correlation with extracellular polysaccharide secreted by Curvibacter sp. strain HJ-1.

Polaromonas eurypsychrophila sp. nov., isolated from an ice core.

A Gram-stain-negative, aerobic, rod-shaped, beige bacterium, strain B717-2T, was isolated from an ice core drilled from Muztagh Glacier on the Tibetan Plateau, China. According to phylogenetic analyses based on 16S rRNA gene sequences, the novel strain was related most closely to Polaromonas vacuolataand shared 97.7 % similarity with the type strain of this species. It grew optimally at pH 7, at 15 °C and with 2 % (w/v) NaCl. Major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), summed feature 8 (C18 : 1ω7c, C18 : 1ω6c) and C16 : 0. The sole respiratory quinone was Q-8. The DNA G+C content was 63.4 mol %. In DNA-DNA hybridization tests, strain B717-2T shared 37.0±1.9, 30.0±1.7, 26.0±0.9, 23.4±0.5 and 18.4±1.9 % DNA-DNA relatedness with Polaromonas jejuensisJS12-13T, P. vacuolata 34-PT, Polaromonas aquatica CCUG 39402T, Polaromonas glacialisCr4-12T and Polaromonas cryoconitiCr4-35T, respectively. Based on the phenotypic, phylogenetic and genetic characteristics, strain B717-2T represents a novel species of the genus Polaromonas, for which the name Polaromonaseurypsychrophila sp. nov. is proposed. The type strain is B717-2T (=CGMCC 1.15322T=JCM 31171T).

An automated Genomes-to-Natural Products platform (GNP) for the discovery of modular natural products.

Bacterial natural products are a diverse and valuable group of small molecules, and genome sequencing indicates that the vast majority remain undiscovered. The prediction of natural product structures from biosynthetic assembly lines can facilitate their discovery, but highly automated, accurate, and integrated systems are required to mine the broad spectrum of sequenced bacterial genomes. Here we present a genome-guided natural products discovery tool to automatically predict, combinatorialize and identify polyketides and nonribosomal peptides from biosynthetic assembly lines using LC-MS/MS data of crude extracts in a high-throughput manner. We detail the directed identification and isolation of six genetically predicted polyketides and nonribosomal peptides using our Genome-to-Natural Products platform. This highly automated, user-friendly programme provides a means of realizing the potential of genetically encoded natural products.

Mercaptosuccinate metabolism in Variovorax paradoxus strain B4--a proteomic approach.

Variovorax paradoxus B4 was isolated due to its ability to degrade the organic thiol compound mercaptosuccinate, which could be a promising precursor for novel polythioesters. The analysis of the proteome of this Gram-negative bacterium revealed several proteins with significantly increased expression during growth of cells with mercaptosuccinate as carbon source when compared to cells grown with gluconate or succinate. Among those, a large number of proteins involved in amino acid metabolism were identified, e.g., adenosylhomocysteinase and glutamate-ammonia ligase. Additionally, detection of superoxide dismutase strengthened the assumption of enhanced stress levels in mercaptosuccinate-grown cells. Several isoforms of a rhodanese domain-containing protein exhibited particularly increased expression during growth with mercaptosuccinate in comparison to gluconate (factor 14.2, stationary phase) or to succinate (factor 15.4, stationary phase). Besides this, augmented expression of the hypothetical protein VAPA_1c41240 raised attention. VAPA_1c41240 exhibited up to 13.3-fold (mercaptosuccinate vs gluconate) or 9.5-fold (mercaptosuccinate vs succinate) increased expression levels, and in silico searches revealed that this protein might be a thiol dioxygenase. Based on these results, a novel degradation pathway is proposed for mercaptosuccinate. The newly identified putative mercaptosuccinate dioxygenase could convert mercaptosuccinate to sulfinosuccinate by the introduction of two molecules of oxygen. Subsequently, sulfinosuccinate would be cleaved into succinate and sulfite either by a yet unknown enzyme, by spontaneous hydrolysis, or by the putative mercaptosuccinate dioxygenase itself. Succinate could then enter the central metabolism, while detoxification of sulfite could be achieved by the previously identified putative molybdopterin oxidoreductase. Biochemical studies will be done in the future to confirm this pathway.

Pelopuradazole, a new imidazole derivative alkaloid from the marine bacteria Pelomonas puraquae sp. nov.

One new imidazole derivative alkaloid pelopuradazole (1), together with three known alkaloids as in 3H-imidazole-4-carboxylic acid (2), 1H-pyrrole-2-carboxylic acid (3) and 2-methyl-3H-imidazole-4-carboxylic acid (4) and two known cyclo-dipeptides pelopurin A (5) and pelopurin B (6), has been isolated from the marine bacterium Pelomonas puraquae sp. nov. Pelopuradazole (1) was a new imidazole derivative alkaloid, while compounds 2, 3, 5 and 6 were firstly obtained as natural products. Compounds 1-6 were isolated from P. puraquae sp. nov. for the first time.

Synchrotron-based chemical nano-tomography of microbial cell-mineral aggregates in their natural, hydrated state.

Chemical nano-tomography of microbial cells in their natural, hydrated state provides direct evidence of metabolic and chemical processes. Cells of the nitrate-reducing Acidovorax sp. strain BoFeN1 were cultured in the presence of ferrous iron. Bacterial reduction of nitrate causes precipitation of Fe(III)-(oxyhydr)oxides in the periplasm and in direct vicinity of the cells. Nanoliter aliquots of cell-suspension were injected into custom-designed sample holders wherein polyimide membranes collapse around the cells by capillary forces. The immobilized, hydrated cells were analyzed by synchrotron-based scanning transmission X-ray microscopy in combination with angle-scan tomography. This approach provides three-dimensional (3D) maps of the chemical species in the sample by employing their intrinsic near-edge X-ray absorption properties. The cells were scanned through the focus of a monochromatic soft X-ray beam at different, chemically specific X-ray energies to acquire projection images of their corresponding X-ray absorbance. Based on these images, chemical composition maps were then calculated. Acquiring projections at different tilt angles allowed for 3D reconstruction of the chemical composition. Our approach allows for 3D chemical mapping of hydrated samples and thus provides direct evidence for the localization of metabolic and chemical processes in situ.