Formulation of biogenic fluorescent pigmented PHB nanoparticles from Rhodanobacter sp. for drug delivery.

Biogenic nanoparticles (NPs) have emerged as promising therapeutic formulations in effective drug delivery. Despite of various positive attributes, these NPs are often conjugated with various cytotoxic organic fluorophores for bioimaging, thereby reducing its effectiveness as a potential carrier. Herein, we aim to formulate biogenic fluorescent pigmented polyhydroxybutyrate (PHB) NPs from Rhodanobacter sp. strain KT31 (OK001852) for drug delivery. The bacterial strain produced 0.5 g L-1 of polyhydroxyalkanoates (PHAs) from 2.04 g L-1 of dry cell weight (DCW) under optimised conditions via submerged fermentation. Further, structural, thermal, and morphological charactersiation of the extracted PHAs was conducted using advance analytical technologies. IR spectra at 1719.25 cm-1 confirmed presence of C = O functional group PHB. NMR and XRD analysis validated the chemical structure and crystallinity of PHB. TG-DTA revealed Tm (168 °C), Td (292 °C), and Xc (35%) of the PHB. FE-SEM imaging indicated rough surface of the PHB film and the biodegradability was confirmed from open windro composting. WST1 assay showed no significant cell death (> 50%) from 100 to 500 µg/mL, endorsing non-cytotoxic nature of PHB. PHB NPs were uniform, smooth and spherical with size distribution and mean zeta potential 44.73 nm and 0.5 mV. IR and XRD peaks obtained at 1721.75 cm-1 and 48.42 Å denoted C = O and crystalline nature of PHB. Cell proliferation rate of PHB NPs was quite significant at 50 µg/mL, establishing the non-cytotoxic nature of NPs. Further, in vitro efficacy of the PHB NPs needs to be evaluated prior to the biomedical applications.

Benchmark and performance of long-range corrected time-dependent density functional tight binding (LC-TD-DFTB) on rhodopsins and light-harvesting complexes.

The chromophores of rhodopsins (Rh) and light-harvesting (LH) complexes still represent a major challenge for a quantum chemical description due to their size and complex electronic structure. Since gradient corrected and hybrid density functional approaches have been shown to fail for these systems, only range-separated functionals seem to be a promising alternative to the more time consuming post-Hartree-Fock approaches. For extended sampling of optical properties, however, even more approximate approaches are required. Recently, a long-range corrected (LC) functional has been implemented into the efficient density functional tight binding (DFTB) method, allowing to sample the excited states properties of chromophores embedded into proteins using quantum mechanical/molecular mechanical (QM/MM) with the time-dependent (TD) DFTB approach. In the present study, we assess the accuracy of LC-TD-DFT and LC-TD-DFTB for rhodopsins (bacteriorhodopsin (bR) and pharaonis phoborhodopsin (ppR)) and LH complexes (light-harvesting complex II (LH2) and Fenna-Matthews-Olson (FMO) complex). This benchmark study shows the improved description of the color tuning parameters compared to standard DFT functionals. In general, LC-TD-DFTB can exhibit a similar performance as the corresponding LC functionals, allowing a reliable description of excited states properties at significantly reduced cost. The two chromophores investigated here pose complementary challenges: while huge sensitivity to external field perturbation (color tuning) and charge transfer excitations are characteristic for the retinal chromophore, the multi-chromophoric character of the LH complexes emphasizes a correct description of inter-chromophore couplings, giving less importance to color tuning. None of the investigated functionals masters both systems simultaneously with satisfactory accuracy. LC-TD-DFTB, at the current stage, although showing a systematic improvement compared to TD-DFTB cannot be recommended for studying color tuning in retinal proteins, similar to some of the LC-DFT functionals, because the response to external fields is still too weak. For sampling of LH-spectra, however, LC-TD-DFTB is a viable tool, allowing to efficiently sample absorption energies, as shown for three different LH complexes. As the calculations indicate, geometry optimization may overestimate the importance of local minima, which may be averaged over when using trajectories. Fast quantum chemical approaches therefore may allow for a direct sampling of spectra in the near future.

Proteomic Analysis Reveals Inflammation Modulation of κ/ι-Carrageenan Hexaoses in Lipopolysaccharide-Induced RAW264.7 Macrophages.

κ/ι-Carrageenan hexaoses (κ/ι-neocarrahexaoses, KCO-4) are a type of carrageenan oligosaccharide that have a broad spectrum of bioactivities due to the presence of sulfate groups. However, the anti-inflammatory capacity of purified carrageenan oligosaccharides and the underlying mechanism has not been completely elucidated. The present study aimed to investigate inflammatory signaling modulation of KCO-4 in LPS-induced macrophages using a quantitative proteomic strategy. KCO-4 inhibited the oversecretion of inflammatory mediators (i.e., NO, TNF-α, IL-1ß, IL-8, iNOS, and COX-2). KCO-4 treatment altered proteome profile, and metabolic processes in mitochondria were significantly disrupted. The IPA network analysis proposed that KCO-4 triggered the NF-κB signaling pathway-dependent anti-inflammation process through the inhibition of CD14/Rel@p50 in LPS-induced RAW264.7 macrophages. These data improve our understanding of the anti-inflammatory mechanism and contribute to exposure biomarker screening of κ-carrageenan oligosaccharides.

Ferrovibrio xuzhouensis sp. nov., a cyhalothrin-degrading bacterium isolated from cyhalothrin contaminated wastewater.

A novel cyhalothrin-degrading strain, designated as LM-6(T), was isolated from a cyhalothrin contaminated wastewater sample. The bacterium was found to be Gram stain-negative, non-spore-forming, vibrio-shaped, and motile with a single polar flagellum. Strain LM-6(T) was observed to grow optimally at 28-30 °C, pH 6.0 and in the absence of NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain LM-6(T) is a member of the genus Ferrovibrio, and showed the highest sequence similarity with Ferrovibrio denitrificans Sp-1(T) (97.7 %), followed by Taonella mepensis H1(T) (93.3 %). The major fatty acids of strain LM-6(T) (>5 %) were determined to be C18:1 ω7c and/or C18:1 ω6c, C16:0, C18:1 2-OH and C17:1 iso I and/or anteiso B. The major polar lipids were identified to be phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmethylethanolamine. The major respiratory quinone was determined to be ubiquinone-10. The genomic DNA G+C content of strain LM-6(T) is 66.5 mol %. Strain LM-6(T) showed low DNA-DNA relatedness with F. denitrificans Sp-1(T) (53.1 ± 0.5 %). On the basis of phylogenetic, genomic, phenotypic and chemotaxonomic data, strain LM-6(T) is considered to represent a novel species of the genus Ferrovibrio, for which the name Ferrovibrio xuzhouensis sp. nov. is proposed. The type strain is Ferrovibrio xuzhouensis LM-6(T) (=KCTC 42182(T) = ACCC 19710(T)).

Magnetotactic bacteria as potential sources of bioproducts.

Magnetotactic bacteria (MTB) produce intracellular organelles called magnetosomes which are magnetic nanoparticles composed of magnetite (Fe3O4) or greigite (Fe3S4) enveloped by a lipid bilayer. The synthesis of a magnetosome is through a genetically controlled process in which the bacterium has control over the composition, direction of crystal growth, and the size and shape of the mineral crystal. As a result of this control, magnetosomes have narrow and uniform size ranges, relatively specific magnetic and crystalline properties, and an enveloping biological membrane. These features are not observed in magnetic particles produced abiotically and thus magnetosomes are of great interest in biotechnology. Most currently described MTB have been isolated from saline or brackish environments and the availability of their genomes has contributed to a better understanding and culturing of these fastidious microorganisms. Moreover, genome sequences have allowed researchers to study genes related to magnetosome production for the synthesis of magnetic particles for use in future commercial and medical applications. Here, we review the current information on the biology of MTB and apply, for the first time, a genome mining strategy on these microorganisms to search for secondary metabolite synthesis genes. More specifically, we discovered that the genome of the cultured MTB Magnetovibrio blakemorei, among other MTB, contains several metabolic pathways for the synthesis of secondary metabolites and other compounds, thereby raising the possibility of the co-production of new bioactive molecules along with magnetosomes by this species.

Statistical approach for the enhanced production of cold-active ß-galactosidase from Thalassospira frigidphilosprofundus: a novel marine psychrophile from deep waters of Bay of Bengal.

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active ß-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for ß-galactosidase production. Under optimal conditions, the production of ß-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. ß-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active ß-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active ß-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.

Thalassospiramide G, a new γ-amino-acid-bearing peptide from the marine bacterium Thalassospira sp.

In the chemical investigation of marine unicellular bacteria, a new peptide, thalassospiramide G (1), along with thalassospiramides A and D (2-3), was discovered from a large culture of Thalassospira sp. The structure of thalassospiramide G, bearing γ-amino acids, such as 4-amino-5-hydroxy-penta-2-enoic acid (AHPEA), 4-amino-3,5-dihydroxy-pentanoic acid (ADPA), and unique 2-amino-1-(1H-indol-3-yl) ethanone (AIEN), was determined via extensive spectroscopic analysis. The absolute configuration of thalassospiramide D (3), including 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), was rigorously determined by 1H-1H coupling constant analysis and chemical derivatization. Thalassospiramides A and D (2-3) inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells, with IC(50) values of 16.4 and 4.8 µM, respectively.

Powering the future of molecular artificial photosynthesis with light-harvesting metallosupramolecular dye assemblies.

Chemical ingenuity will play a significant role in solving the greatest challenge currently facing society: providing clean and carbon neutral energy for all of humanity. Molecular artificial photosynthesis is an emerging technology based on principles learned from Nature where individual components perform the essential light-harvesting, charge-separation, and water splitting functions to store solar energy in the form of chemical bonds. This tutorial review focuses specifically on the application of metallosupramolecular self-assembly strategies to interface solar fuel catalysts with photosensitizers and construct light-harvesting antennae capable of achieving panchromatic absorption and directional energy concentration.

Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria.

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus.

Conformational properties of two exopolysaccharides produced by Inquilinus limosus, a cystic fibrosis lung pathogen.

Inquilinus limosus is a multi-resistant bacterium found in the respiratory tract of patients with cystic fibrosis. This bacterium produces two unique fully pyruvylated exopolysaccharides in similar quantities: an α-(1→2)-linked mannan and a ß-(1→3)-linked glucan. We employed molecular modelling methods to probe the characteristic conformations and dynamics of these polysaccharides, with corroboration from potentiometric titrations and circular dichroism experiments. Our calculations reveal different structural motifs for the mannan and glucan polysaccharides: the glucan forms primarily right-handed helices with a wide range of extensions, while the mannan forms only left-handed helices. This finding is supported by our circular dichroism experiments. Our calculations also show that the (1→3)-ß-d-Glcp linkage is more dynamically flexible than the (1→2)-α-d-Manp: the glucan characteristically forms a range of wide helices with large central cavities. In contrast, the mannan forms rigid regular 'bottlebrush' helices with a minimal central cavity. The widely different character of these two polymers suggests a possible differentiation of biological roles.

Genus specific unusual carotenoids in purple bacteria, Phaeospirillum and Roseospira: structures and biosyntheses.

Phototrophic bacteria necessarily contain carotenoids for photosynthesis, and a few phototrophic purple bacteria accumulate unusual carotenoids. The carotenoids in the genera Phaeospirillum and Roseospira were identified using spectroscopic methods. All species of the genus Phaeospirillum contained characteristic polar carotenoids in addition to lycopene and hydroxylycopene (rhodopin); hydroxylycopene glucoside, dihydroxylycopene, and its mono- and/or diglucosides. From the structures of these carotenoids, their accumulation was suggested to be due to absence of CrtD (acyclic carotenoid C-3,4 desaturase) and to possession of glucosyltransferase. Species of the genus Roseospira have been reported to have unusual absorption spectra in acetone extract, and they were found to accumulate 3,4-didehydrorhodopin as a major carotenoid. This may be due to low activity of CrtF (acyclic 1-hydroxycarotenoid methyltransferase). The study concludes in identifying genus specific unusual carotenoids, which is probably due to characteristic nature of some carotenogenesis enzymes.

Effects of ethanol, formaldehyde, and gentle heat fixation in confocal resonance Raman microscopy of purple nonsulfur bacteria.

Resonance Raman microscopy is well suited to examine living bacterial samples without further preparation. Therefore, comparatively little thought has been given to its compatibility with common fixation methods. However, fixation of cell samples is a very important tool in the microbiological sciences, allowing the preservation of samples in a specific condition for further examination, future measurements, transport, or later reference. We examined the effects of three common fixatives-ethanol, formaldehyde solution, and gentle heat--on the resonant Raman spectrum of three generic bacteria species, Rhodobacter sphaeroides DSM 158(T), Rhodopseudomonas palustris DSM 123(T), and Rhodospirillum rubrum DSM 467(T), holding carotenoid- and heme-chromophores in confocal Raman microscopy. In addition, we analyzed the effect of poly-L-lysine coating of microscope slides, widely used for mounting biological and medical samples, on subsequent confocal Raman measurements of native and fixed samples. The results indicate that ethanol is preferable to formaldehyde as fixative if applied for less than 24 h, whereas heat fixation has a strong, detrimental effect on the resonant Raman spectrum of bacteria. Formaldehyde fixation excels at fixation times above 24 h, but causes an overall reduction in signal intensity. Poly-L-lysine coating has no discernable effect on the Raman spectra of samples fixed with ethanol or heat, but it further decreases the signal intensity, especially at higher wavenumbers, in the spectra of samples fixed with formaldehyde.

Phaeospirillum chandramohanii sp. nov., a phototrophic alphaproteobacterium with carotenoid glycosides.

A Gram-negative, spiral-shaped, phototrophic, purple non-sulfur bacterial strain, designated JA145T, was isolated from a freshwater habitat. Cells of strain JA145T were motile by means of a monopolar flagellum. Intracellular photosynthetic membranes were of the stacked type. Bacteriochlorophyll a and the carotenoid lycopene and its glucosides were present as photosynthetic pigments. There was no vitamin requirement for strain JA145T. The predominant cellular fatty acids were C16:1omega7c/C16:1omega6c (22.24%), C16:0 (22.97%) and C18:1omega7c (43.24%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JA145T clustered with species of the genus Phaeospirillum, in the class Alphaproteobacteria. The highest sequence similarities of strain JA145T were found with the type strains of Phaeospirillum fulvum (96.12%) and Phaeospirillum molischianum (96.19%). Based on the 16S rRNA gene sequence analysis and the morphological and physiological characteristics, strain JA145T is considered to represent a novel species, for which the name Phaeospirillum chandramohanii sp. nov. is proposed. The type strain is JA145T (=JCM 14933T=KCTC 5703T=NBRC 104961T).

Inhibition of cathelicidin activity by bacterial exopolysaccharides.

The interaction of bacterial exopolysaccharides, produced by opportunistic lung pathogens, with antimicrobial peptides of the innate primate immune system was investigated. The exopolysaccharides were produced by Pseudomonas aeruginosa, Inquilinus limosus and clinical isolates of the Burkholderia cepacia complex, bacteria that are all involved in lung infections of cystic fibrosis patients. The effects of the biological activities of three orthologous cathelicidins from Homo sapiens sapiens, Pongo pygmaeus (orangutan) and Presbitys obscurus (dusky leaf monkey) were examined. Inhibition of the antimicrobial activity of peptides was assessed using minimum inhibitory concentration assays on a reference Escherichia coli strain in the presence and absence of exopolysaccharides, whereas complex formation between peptides and exopolysaccharides was investigated by means of circular dichroism, fluorescence spectroscopy and atomic force microscopy. Biological assays revealed that the higher the negative charge of exopolysaccharides the stronger was their inhibiting effect. Spectroscopic studies indicated the formation of molecular complexes of varying stability between peptides and exopolysaccharides, explaining the inhibition. Atomic force microscopy provided a direct visualization of the molecular complexes. A model is proposed where peptides with an alpha-helical conformation interact with exopolysaccharides through electrostatic and other non-covalent interactions.

[Comparative study of the fatty acid composition of some groups of purple nonsulfur bacteria].

The fatty acid composition (FAC) of 43 strains of purple nonsulfur bacteria belonging to six genera--Rubrivivax, Rhodopseudomonas, Rhodoplanes, Blastochloris, Rhodobium, and Rhodomicrobium--was studied by capillary gas chromatography. The cultures were grown on standard medium under standard conditions. Automatic identification of the fatty acid methyl esters and statistical processing of the results were performed by the computerized Microbial Identification System (M.I.S). Significant differences between the FACs of different genera, species, and, sometimes, strains were revealed. 16S rRNA genes of some of the new isolates, primarily those having a specific FAC, were sequenced. The taxonomic status of a number of the strains in question was determined using the FAC characteristics as one of the criteria. It was shown that the FAC characteristics may be used both for affiliating the isolates to known species and for revealing new taxa.

Exopolysaccharides produced by Inquilinus limosus, a new pathogen of cystic fibrosis patients: novel structures with usual components.

The major cause of morbidity and mortality in patients with cystic fibrosis, an autosomal recessive disorder, is chronic microbial colonisation of the major airways that leads to exacerbation of pulmonary infection. Several different microbes colonise cystic fibrosis lungs, and Pseudomonas aeruginosa is one of the most threatening, since the establishment of mucoid (alginate producing) strains is ultimately associated with the patient's death. Very recently a new bacterium, named Inquilinus limosus, was repeatedly found infecting the respiratory tract of cystic fibrosis patients. Its multi-resistance characteristic to antibiotics might result in the spreading of I. limosus infection among the cystic fibrosis community, as recently happened with strains of the Burkholderia cepacia complex. Since exopolysaccharides are recognised as important virulence factors in lung infections, the primary structure of the polysaccharide produced by I. limosus strain LMG 20952(T) was investigated as the first step in understanding its role in pathogenesis. The structure was determined by means of methylation analysis, acid degradations, mass spectrometry and NMR spectroscopy. The results showed that the bacterium produced a mixture constituted of the following polymers: [3)-[4,6-O-(1-carboxyethylidene)]-beta-D-Glcp(1-->]n; [2)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Manp(1-->]n. Both polymers were completely substituted with pyruvyl ketal groups, a novel structural characteristic not previously found in bacterial polysaccharides. The absolute configuration of all pyruvyl groups was S. Inspection of possible local conformations assumed by the two polysaccharide chains showed features, which might provide interesting clues for understanding structure-function relationships.

Skermanella aerolata sp. nov., isolated from air, and emended description of the genus Skermanella.

A light-pink-coloured bacterium, designated strain 5416T-32(T), was isolated from an air sample in Korea. Cells of the strain were strictly aerobic, Gram-negative, motile (single polar or subpolar flagellum) and rod-shaped. Optimal growth occurred at 25-30 degrees C and at pH 6.0-7.0. The major quinones were Q-10 and Q-8. The major fatty acids were C(18 : 1) omega 7c (53.8 %) and C(16 : 0) (15.9 %). The G+C content of the genomic DNA was 65.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 5416T-32(T) was most closely related to Skermanella parooensis, with a similarity of 96.2 %, but relatively low sequence similarities (<92 %) were found with respect to other species with validly published names held in GenBank. Phenotypic and genotypic analyses indicated that strain 5416T-32(T) could not be assigned to any recognized species. Therefore strain 5416T-32(T) represents a novel species of the genus Skermanella, for which the name Skermanella aerolata sp. nov. is proposed. The type strain is 5416T-32(T) (=KACC 11604(T)=DSM 18479(T)).

Thalassospiramides A and B, immunosuppressive peptides from the marine bacterium Thalassospira sp.

[structure: see text] Two new cyclic peptides, thalassospiramides A and B (1 and 2), were isolated from a new member of the marine alpha-proteobacterium Thalassospira. The thalassospiramides, the structures of which were assigned by combined spectral and chemical methods, bear unusual gamma-amino acids and show immunosuppressive activity in an interleukin-5 production inhibition assay (IC50 = 5 muM for thalassospiramide B).

An acidic protein aligns magnetosomes along a filamentous structure in magnetotactic bacteria.

Magnetotactic bacteria are widespread aquatic microorganisms that use unique intracellular organelles to navigate along the Earth's magnetic field. These organelles, called magnetosomes, consist of membrane-enclosed magnetite crystals that are thought to help to direct bacterial swimming towards growth-favouring microoxic zones at the bottom of natural waters. Questions in the study of magnetosome formation include understanding the factors governing the size and redox-controlled synthesis of the nano-sized magnetosomes and their assembly into a regular chain in order to achieve the maximum possible magnetic moment, against the physical tendency of magnetosome agglomeration. A deeper understanding of these mechanisms is expected from studying the genes present in the identified chromosomal 'magnetosome island', for which the connection with magnetosome synthesis has become evident. Here we use gene deletion in Magnetospirillum gryphiswaldense to show that magnetosome alignment is coupled to the presence of the mamJ gene product. MamJ is an acidic protein associated with a novel filamentous structure, as revealed by fluorescence microscopy and cryo-electron tomography. We suggest a mechanism in which MamJ interacts with the magnetosome surface as well as with a cytoskeleton-like structure. According to our hypothesis, magnetosome architecture represents one of the highest structural levels achieved in prokaryotic cells.