Living in a bottle: Bacteria from sediment-associated Mediterranean waste and potential growth on polyethylene terephthalate.

Ocean pollution is a worldwide environmental challenge that could be partially tackled through microbial applications. To shed light on the diversity and applications of the bacterial communities that inhabit the sediments trapped in artificial containers, we analyzed residues (polyethylene terephthalate [PET] bottles and aluminum cans) collected from the Mediterranean Sea by scanning electron microscopy and next generation sequencing. Moreover, we set a collection of culturable bacteria from the plastisphere that were screened for their ability to use PET as a carbon source. Our results reveal that Proteobacteria are the predominant phylum in all the samples and that Rhodobacteraceae, Woeseia, Actinomarinales, or Vibrio are also abundant in these residues. Moreover, we identified marine isolates with enhanced growth in the presence of PET: Aquimarina intermedia, Citricoccus spp., and Micrococcus spp. Our results suggest that the marine environment is a source of biotechnologically promising bacterial isolates that may use PET or PET additives as carbon sources.

Streptacidiphilus fuscans sp. nov., a novel actinobacterium isolated from the root of pumpkin (Cucurbita moschata).

A novel acidophilic actinobacterium, designated strain NEAU-YB345T, was isolated from a pumpkin root collected from Mudanjiang, Heilongjiang Province, northeast PR China. Based on 16S rRNA gene sequence similarity and chemotaxonomic and morphological properties, the isolate was assigned to the genus Streptacidiphilus, with the high 16S rRNA gene sequence similarities to Streptacidiphilus melanogenes JCM 16224T (99.2 %), Streptacidiphilus anmyonensis JCM 16223T (99.1 %) and Streptacidiphilus jiangxiensis JCM 12277T (98.7 %). Its cell wall contained ll-diaminopimelic acid as the major diamino acid. Rhamnose, ribose, glucose and galactose were the detected sugars from the whole-cell hydrolysates. The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified phospholipid. The menaquinones were MK-9(H8) and MK-9(H6). Major fatty acids were C16 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C15 : 0. Phylogenetic analysis using 16S rRNA gene and whole-genome sequences placed the strain in distinct clades but within the genus Streptacidiphilus. The DNA G+C content was 71.2 mol%. Based on DNA-DNA relatedness and physiological and biochemical data, the isolate could be distinguished from its closest relatives. Therefore, strain NEAU-YB345T represents a novel species of the genus Streptacidiphilus, for which the name Streptacidiphilus fuscans sp. nov. is proposed. The type strain is NEAU-YB345T (=CCTCC AA 2020030T=JCM 33976T).

Actinomycetes Enrich Soil Rhizosphere and Improve Seed Quality as well as Productivity of Legumes by Boosting Nitrogen Availability and Metabolism.

The use of actinomycetes for improving soil fertility and plant production is an attractive strategy for developing sustainable agricultural systems due to their effectiveness, eco-friendliness, and low production cost. Out of 17 species isolated from the soil rhizosphere of legume crops, 4 bioactive isolates were selected and their impact on 5 legumes: soybean, kidney bean, chickpea, lentil, and pea were evaluated. According to the morphological and molecular identification, these isolates belong to the genus Streptomyces. Here, we showed that these isolates increased soil nutrients and organic matter content and improved soil microbial populations. At the plant level, soil enrichment with actinomycetes increased photosynthetic reactions and eventually increased legume yield. Actinomycetes also increased nitrogen availability in soil and legume tissue and seeds, which induced the activity of key nitrogen metabolizing enzymes, e.g., glutamine synthetase, glutamate synthase, and nitrate reductase. In addition to increased nitrogen-containing amino acids levels, we also report high sugar, organic acids, and fatty acids as well as antioxidant phenolics, mineral, and vitamins levels in actinomycete treated legume seeds, which in turn improved their seed quality. Overall, this study shed the light on the impact of actinomycetes on enhancing the quality and productivity of legume crops by boosting the bioactive primary and secondary metabolites. Moreover, our findings emphasize the positive role of actinomycetes in improving the soil by enriching its microbial population. Therefore, our data reinforce the usage of actinomycetes as biofertilizers to provide sustainable food production and achieve biosafety.

Structure of the Dietzia Mrp complex reveals molecular mechanism of this giant bacterial sodium proton pump.

Multiple resistance and pH adaptation (Mrp) complexes are sophisticated cation/proton exchangers found in a vast variety of alkaliphilic and/or halophilic microorganisms, and are critical for their survival in highly challenging environments. This family of antiporters is likely to represent the ancestor of cation pumps found in many redox-driven transporter complexes, including the complex I of the respiratory chain. Here, we present the three-dimensional structure of the Mrp complex from a Dietzia sp. strain solved at 3.0-Å resolution using the single-particle cryoelectron microscopy method. Our structure-based mutagenesis and functional analyses suggest that the substrate translocation pathways for the driving substance protons and the substrate sodium ions are separated in two modules and that symmetry-restrained conformational change underlies the functional cycle of the transporter. Our findings shed light on mechanisms of redox-driven primary active transporters, and explain how driving substances of different electric charges may drive similar transport processes.

Discovery of ANTAR-RNAs and their Mechanism of Action in Mycobacteria.

Non-coding RNAs play pivotal roles in bacterial signaling. However, RNAs from certain phyla (specially high-GC actinobacteria) still remain elusive. Here, by re-engineering the existing genome-wide search approach, we discover a family of structurally conserved RNAs that are present ubiquitously across actinobacteria, including mycobacteria. In vitro analysis shows that RNAs belonging to this family bind response-regulator proteins that contain the widely prevalent ANTAR domain. The Mycobacterium tuberculosis ANTAR protein gets phosphorylated by a histidine kinase and interacts with RNA only in its phosphorylated state. These newly identified RNAs reside only in certain transcripts and typically overlap with the ribosome-binding site, regulating translation of these transcripts. In this way, the RNAs directly link signaling pathways to translational control, thus expanding the mechanistic tool kit available for ANTAR-based control of gene expression. In mycobacteria, we find that RNAs targeted by ANTAR proteins majorly encode enzymes of lipid metabolism and associated redox pathways. This now allows us to identify the key genes that mediate ANTAR-dependent control of lipid metabolism. Our study establishes the identity and wide prevalence of ANTAR-target RNAs in mycobacteria, bringing RNA-mediated regulation in these bacteria to the center stage.

Molecular Basis of Mannose Recognition by Pradimicins and their Application to Microbial Cell Surface Imaging.

Naturally occurring pradimicins (PRMs) show specific recognition of d-mannose (d-Man) in aqueous media, which has never been achieved by artificial small molecules. Although the Ca2+-mediated dimerization of PRMs is essential for their d-Man binding, the dimeric structure has yet to be elucidated, leaving the question open as to how PRMs recognize d-Man. Thus, we herein report the structural elucidation of the dimer by a combination of X-ray crystallography and solid-state NMR spectroscopy. Coupled with our previous knowledge regarding the d-Man binding geometry of PRMs, elucidation of the dimer allowed reliable estimation of the mode of d-Man binding. Based on the binding model, we further developed an azide-functionalized PRM derivative (PRM-Azide) with d-Man binding specificity. Notably, PRM-Azide stained Candida rugosa cells having mannans on their cell surface through conjugation with the tetramethylrhodamine fluorophore. The present study provides the practical demonstration that PRMs can serve as lectin mimics for use in glycobiological studies.

Effects of Different Carbon Sources on Enzyme Production and Ultrastructure of Cellulosimicrobium cellulans.

The secretomes of the strain Cellulosimicrobium cellulans F16 grown on different carbon sources were analyzed by zymography, and the subcellular surface structures were extensively studied by electron microscope. The exo-cellulase and xylanase systems were sparse when cells were grown on soluble oligosaccharides, but were significantly increased when grown on complex and water-insoluble polysaccharides, such as Avicel, corn cob, and birchwood xylan. The cellulosome-like protuberant structures were clearly observed on the cell surfaces of strain F16 grown on cellulose, with diameters of 15-20 nm. Fibrous structures that connected the adjacent cells can be seen under microscope. Moreover, protuberances that adsorbed the cell to cellulose were also observed. As the adhesion of Cellulosimicrobium cellulans cells onto cellulose surfaces occurs via thick bacterial curdlan-type exopolysaccharides (EPS), such surface layer is potentially important in the digestion of insoluble substrates such as cellulose or hemicellulose, and the previously reported xylanosomes are part of such complex glycocalyx layer on the surface of the bacterial cell.

Yellow coloured mats from lava tubes of La Palma (Canary Islands, Spain) are dominated by metabolically active Actinobacteria.

Microbial diversity in lava tubes from Canary Islands (Spain) has never been explored thus far offering a unique opportunity to study subsurface microbiology. Abundant yellow coloured mats developing on coralloid speleothems in a lava tube from La Palma Islands were studied by next-generation sequencing and DNA/RNA clone library analyses for investigating both total and metabolically active bacteria. In addition, morphological and mineralogical characterization was performed by field emission scanning electron microscopy (FESEM), micro-computed tomography, X-ray diffraction and infrared spectroscopy to contextualize sequence data. This approach showed that the coralloid speleothems consist of banded siliceous stalactites composed of opal-A and hydrated halloysite. Analytical pyrolysis was also conducted to infer the possible origin of cave wall pigmentation, revealing that lignin degradation compounds can contribute to speleothem colour. Our RNA-based study showed for the first time that members of the phylum Actinobacteria, with 55% of the clones belonging to Euzebyales order, were metabolically active components of yellow mats. In contrast, the DNA clone library revealed that around 45% of clones were affiliated to Proteobacteria. Composition of microbial phyla obtained by NGS reinforced the DNA clone library data at the phylum level, in which Proteobacteria was the most abundant phylum followed by Actinobacteria.

Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.

Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.

Microencapsulation of Gulosibacter molinativorax ON4T cells by a spray-drying process using different biopolymers.

Molinate is a thiocarbamate herbicide used in rice crop protection. As other pesticides, molinate is a recognized environmental pollutant and bio-accumulated by some wildlife forms. Gulosibacter molinativorax ON4T is able to hydrolyse molinate into metabolites which are further degraded by other un-related bacteria. Hence, it can be used in molinate bioremediation processes. The aim of this work was to investigate the possibility of producing G. molinativorax ON4T microparticles, using different non-toxic biopolymers (arabic gum, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. Several formulations of microparticles were prepared, and their physicochemical structures were analyzed by scanning electron microscopy (SEM), laser granulometry analysis and zeta potential analysis. The obtained microparticles were evaluated considering their ability to degrade molinate, the metabolic activity (by colour development of the tetrazolium violet redox), and also the survival rate and shelf-life/storage stability of microparticles. Based on their molinate degrading activity, the biopolymers calcium alginate and modified chitosan cross-linked with tripolyphosphate appear to be the best options for the microencapsulation of the G. molinativorax ON4T. However, the microparticles produced with modified chitosan cross-linked with tripolyphosphate present the best combination of physical properties and activity degradation of molinate.

Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82.

L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia. A newly isolated actinomycetes strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the GenBank database under accession number KJ467538. L-asparaginase was purified from the crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified enzyme were carried out. The optimum pH, temperature and incubation time for maximum L-asparaginase activity were found to be 8.5, 40 °C and 30 min, respectively. The optimum substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were 0.01007 M and 95.08 Uml(-1)min(-1), respectively. The half-life time (T1/2) was 184.91 min at 50 °Ð¡, while being 179.53 min at 60 °Ð¡. The molecular weight of the subunits of L-asparaginase was found to be approximately 53 kDa by SDS-PAGE analysis. The purified L-asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-fold. The present work for the first time reported more information in the production, purification and characterization of L-asparaginase produced by newly isolated actinomycetes Streptomyces fradiae NEAE-82.

Streptosporangium lutulentum sp. nov., Streptosporangium fenghuangense sp. nov. and Streptosporangium corydalis sp. nov., three novel actinobacterial species isolated from National Forest Park of Fenghuang Mountain.

Three novel actinobacteria, designated strains NEAU-FSHN1(T), NEAU-hd-3(T) and NEAU-Y6(T), were isolated from a stream base, soil adjacent to the stream and a root of Corydalis yanhusuo L, respectively, collected from Wuchang, Heilongjiang Province, China. A polyphasic study was carried out to establish the taxonomic positions of these strains. The three strains were observed to form scant aerial hyphae that differentiated into spherical spore vesicles. The phylogenetic analysis based on the 16S rRNA gene sequences of strains NEAU-FHSN1(T), NEAU-hd-3(T) and NEAU-Y6(T) showed that the three novel isolates exhibit 99.2 % (NEAU-FHSN1(T)/NEAU-hd-3(T)), 99.2 % (NEAU-FHSN1(T)/NEAU-Y6(T)) and 99.7 % (NEAU-hd-3(T)/NEAU-Y6(T)) 16S rRNA gene sequence similarities with each other and that they are closely related to strains Streptosporangium shengliense NEAU-GH7(T) (sequence similarities 98.72, 98.85, 98.99 %), Streptosporangium roseum DSM 43021(T) (98.65, 98.51, 98.58 %) and Streptosporangium album DSM 43023(T) (98.41, 98.96, 98.89 %). However, the DNA-DNA hybridization values between strains NEAU-FSHN1(T), NEAU-hd-3(T) and NEAU-Y6(T) were 61.2 % (NEAU-FSHN1(T)/NEAU-hd-3(T)), 63.5 % (NEAU-FHSN1(T)/NEAU-Y6(T)) and 65.8 % (NEAU-hd-3(T)/NEAU-Y6(T)), and the values between the three strains and their close phylogenetic relatives were also below 70 %. With reference to phenotypic characteristics, phylogenetic data and DNA-DNA hybridization results, the three strains can be distinguished from each other and their close phylogenetic relatives. Thus, strains NEAU-FHSN1(T), NEAU-hd-3(T) and NEAU-Y6(T) are concluded to represent three novel species of the genus Streptosporangium, for which the names Streptosporangium lutulentum sp. nov., Streptosporangium fenghuangense sp. nov. and Streptosporangium corydalis sp. nov. are proposed. The type strains are NEAU-FHSN1(T) (=CGMCC 4.7141(T) = DSM 46740(T)), NEAU-Y6(T) (=CGMCC 4.7150(T) = DSM 46722(T)) and NEAU-hd3(T) (CGMCC 4.7212(T) = JCM 30058(T)), respectively.

A fusant of Amycolatopsis sp. M3-1 and Pseudomonas sp. Nai8 with high capacity of degrading novel pyrimidynyloxybenzoic herbicide ZJ0273 and naphthalene.

ZJ0273 (propyl 4-(2-(4, 6-demethoxy pyrimidin-2-yloxy) benzylamino) benzoate) is a novel pyrimidynyloxybenzoic-based herbicide developed in China for oilseed crop. This study was aimed to construct new strains capable of degrading naphthalene and ZJ0273 by protoplast fusion between Amycolatopsis sp. M3-1 and Pseudomonas sp. Nai8. Eight recombinant strains were successfully produced, and the strains could simultaneously utilize ZJ0273 and naphthalene as the sole carbon and energy source, respectively. One of recombinant strains, MN6 with higher degrading efficiency, was chosen for further study. Under the condition of pH 7.0, 30 °C, ZJ0273 and naphthalene degradation percent by the recombinant strain MN6 could reach 65.10% (20 days) and 88.46% (48 h), respectively. According to the identified six metabolites (M1-M6) by LC-MS/MS, biodegradation pathway of ZJ0273 was proposed. ZJ0273 biodegradation catalyzed by the recombinant strain MN6 involved continuous biocatalytic reactions such as de-estering, hydrolysis, acylation, C-N cleavage, de-methyl, and ether cleavage reactions.

Microbispora sp. LGMB259 endophytic actinomycete isolated from Vochysia divergens (Pantanal, Brazil) producing ß-carbolines and indoles with biological activity.

Endophytic actinomycetes encompass bacterial groups that are well known for the production of a diverse range of secondary metabolites. Vochysia divergens is a medicinal plant, common in the "Pantanal" region (Brazil) and was focus of many investigations, but never regarding its community of endophytic symbionts. During a screening program, an endophytic strain isolated from the V. divergens, was investigated for its potential to show biological activity. The strain was characterized as Microbispora sp. LGMB259 by spore morphology and molecular analyze using nucleotide sequence of the 16S rRNA gene. Strain LGMB259 was cultivated in R5A medium producing metabolites with significant antibacterial activity. The strain produced 4 chemically related ß-carbolines, and 3 Indoles. Compound 1-vinyl-ß-carboline-3-carboxylic acid displayed potent activity against the Gram-positive bacterial strains Micrococcus luteus NRRL B-2618 and Kocuria rosea B-1106, and was highly active against two human cancer cell lines, namely the prostate cancer cell line PC3 and the non-small-cell lung carcinoma cell line A549, with IC50 values of 9.45 and 24.67 µM, respectively. 1-Vinyl-ß-carboline-3-carboxylic acid also showed moderate activity against the yeast Saccharomyces cerevisiae ATCC204508, as well as the phytopathogenic fungi Phyllosticta citricarpa LGMB06 and Colletotrichum gloeosporioides FDC83.

Use of combined microscopic and spectroscopic techniques to reveal interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone.

Although uranium (U) is naturally found in the environment, soil remediation programs will become increasingly important in light of certain human activities. This work aimed to identify U(VI) detoxification mechanisms employed by a bacteria strain isolated from a Chernobyl soil sample, and to distinguish its active from passive mechanisms of interaction. The ability of the Microbacterium sp. A9 strain to remove U(VI) from aqueous solutions at 4 °C and 25 °C was evaluated, as well as its survival capacity upon U(VI) exposure. The subcellular localisation of U was determined by TEM/EDX microscopy, while functional groups involved in the interaction with U were further evaluated by FTIR; finally, the speciation of U was analysed by TRLFS. We have revealed, for the first time, an active mechanism promoting metal efflux from the cells, during the early steps following U(VI) exposure at 25 °C. The Microbacterium sp. A9 strain also stores U intracellularly, as needle-like structures that have been identified as an autunite group mineral. Taken together, our results demonstrate that this strain exhibits a high U(VI) tolerance based on multiple detoxification mechanisms. These findings support the potential role of the genus Microbacterium in the remediation of aqueous environments contaminated with U(VI) under aerobic conditions.

Scanning electron microscopy of bacteria Tetrasphaera duodecadis.

This study reports the characterization of the Tetrasphaera duodecadis bacteria and the techniques used therein. In order to evaluate the morphological characteristics of the T. duodecadis bacteria scanning electron microscope (SEM) was used throughout its different growth stages. These microorganisms were grown in vitamin B12 broths with 1% tryptone, 0.2% yeast extract, and 0.1% glucose. The turbidimetric method was employed for the determination of bacterial concentration and growth curve. The SEM results show small agglomerates of 0.8 ± 0.05 µm during the lag phase, and rod-like shapes during the exponential phase with similar shapes in the stationary phase.

A rapid and improved technique for scanning electron microscopy of actinomycetes.

A simple and rapid method for the preparation of actinomycete cultures before scanning electron microscopy (SEM) is reported. Streptomyces strains were grown in starch casein broth with sterile glass wool as support. The cultures were dehydrated by lyophilization eliminating the use of chemical fixatives and dehydrating agents.

Structure and biological properties of lentztrehalose: a novel trehalose analog.

A new trehalose analog, lentztrehalose [4-O-(2,3-dihydroxy-3-methylbutyl)trehalose], was isolated from an actinomycete Lentzea sp. Lentztrehalose is only weakly hydrolyzed by the trehalose-hydrolyzing enzyme, trehalase, so can be regarded as an enzyme-stable analog of trehalose. Although lentztrehalose does not show apparent toxicity to mammalian cells and microbes, it has antitumor activity in mice bearing S-180 sarcoma and Ehrlich carcinoma cells. In ovariectomized mice, lentztrehalose displayed a bone reinforcement effect in the femur that was superior to trehalose and induced non-morbid suppression of weight gain comparable with trehalose. These results indicate that enzyme-stable analogs of trehalose, such as lentztrehalose, may be more beneficial for human health and thus have potential as substitutes for trehalose as a sweetener.

Bacterial epibionts and endolichenic actinobacteria and fungi in the Lower Devonian lichen Chlorolichenomycites salopensis.

The charcoalified fragment of the dorsiventrally organized, internally stratified presumed green algal lichen Chlorolichenomycites salopensis from the Lower Devonian Lochkovian strata in the Welsh Borderland carries bacterial colonies on the upper surface, i.e. the cortex, and actinobacterial filaments in the medulla underneath the photobiont layer. Moreover relatively thin hyphae of presumed endolichenic fungi were found. As in extant lichens, which are best regarded as consortia with an unknown number of participants, this internally stratified, fossil thallus fragment of a presumed green algal lichen harbours a diverse microbial community.

'Candidatus Ancillula trichonymphae', a novel lineage of endosymbiotic Actinobacteria in termite gut flagellates of the genus Trichonympha.

Termite gut flagellates are colonized by host-specific lineages of ectosymbiotic and endosymbiotic bacteria. Previous studies have shown that flagellates of the genus Trichonympha may harbour more than one type of symbiont. Using a comprehensive approach that combined cloning of SSU rRNA genes with fluorescence in situ hybridization and electron microscopy, we investigated the phylogeny and subcellular locations of the symbionts in a variety of Trichonympha species from different termites. The flagellates in Trichonympha Cluster I were the only species associated with 'Endomicrobia', which were located in the posterior part of the cell, confirming previous results. Trichonympha species of Cluster II from the termite genus Incisitermes (family Kalotermitidae) lacked 'Endomicrobia' and were associated with endosymbiotic Actinobacteria, which is highly unusual. The endosymbionts, for which we suggest the name 'Candidatus Ancillula trichonymphae', represent a novel, deep-branching lineage in the Micrococcineae that consists exclusively of clones from termite guts. They preferentially colonized the anterior part of the flagellate host and were highly abundant in all species of Trichonympha Cluster II except Trichonympha globulosa. Here, they were outnumbered by a Desulfovibrio species associated with the cytoplasmic lamellae at the anterior cell pole. Such symbionts are present in both Trichonympha clusters, but not in all species. Unlike the intracellular location reported for the Desulfovibrio symbionts of Trichonympha agilis (Cluster I), the Desulfovibrio symbionts of T. globulosa (Cluster II) were situated in deep invaginations of the plasma membrane that were clearly connected to the exterior of the host cell.