Functionalization of silk with actinomycins from Streptomyces anulatus BV365 for biomedical applications.

Silk, traditionally acclaimed as the "queen of fiber," has been widely used thanks to its brilliant performance such as gentleness, smoothness and comfortableness. Owing to its mechanical characteristics and biocompatibility silk has a definitive role in biomedical applications, both as fibroin and fabric. In this work, the simultaneous dyeing and functionalization of silk fabric with pigments from Streptomyces anulatus BV365 were investigated. This strain produced high amounts of orange extracellular pigments on mannitol-soy flour agar, identified as actinomycin D, C2 and C3. The application of purified actinomycins in the dyeing of multifiber fabric was assessed. Actinomycins exhibited a high affinity towards protein fibers (silk and wool), but washing durability was maintained only with silk. Acidic condition (pH5) and high temperature (65°C) facilitated the silk dyeing. The morphologies and chemical components of the treated silk fabrics were analyzed using scanning electron microscopy and Fourier transform infrared spectroscopy. The results showed the pigments bind to the silk through interaction with the carbonyl group in silk fibroin rendering the functionalized, yet surface that does not cause skin irritation. The treated silk exhibited a remarkable antibacterial effect, while the biocompatibility test performed with 3D-reconstructed human epidermis model indicated safe biological properties, paving the way for future application of this material in medicine.

Discovery and biosynthesis of non-canonical C16-terpenoids from Pseudomonas.

Biosynthesis of sodorifen with a unique C16-bicyclo[3.2.1]octene framework requires an S-adenosyl methionine-dependent methyltransferase SodC and terpene cyclase SodD. While bioinformatic analyses reveal a wide distribution of the sodCD genes organization in bacteria, their functional diversity remains largely unknown. Herein, two sodorifen-type gene clusters, pcch and pcau, from Pseudomonas sp. are heterologously expressed in Escherichia coli, leading to the discovery of two C16 terpenoids. Enzymatic synthesis of these compounds is achieved using the two (SodCD-like) pathway-specific enzymes. Enzyme assays using different combinations of methyltransferases and terpene synthases across the pcch, pcau, and sod pathways reveal a unifying biosynthetic mechanism: all three SodC-like enzymes methylate farnesyl pyrophosphate (FPP) with subsequent cyclization to a common intermediate, pre-sodorifen pyrophosphate. Structural diversification of this joint precursor solely occurs by the subsequently acting individual terpene synthases. Our findings expand basic biosynthetic understanding and structural diversity of unusual C16-terpenoids.

Final Destination? Pinpointing Hyella disjuncta sp. nov. PCC 6712 (Cyanobacteria) Based on Taxonomic Aspects, Multicellularity, Nitrogen Fixation and Biosynthetic Gene Clusters.

Unicellular cyanobacteria inhabit a wide range of ecosytems and can be found throughout the phylum offering space for taxonomic confusion. One example is strain PCC 6712 that was described as Chlorogloea sp. (Nostocales) and later assigned to the genus Chroococcidiopsis (Chroococcidiopsidales). We now show that this strain belongs to the order Pleurocapsales and term it Hyella disjuncta based on morphology, genome analyses and 16S-23S ITS rRNA phylogeny. Genomic analysis indicated that H. disjuncta PCC 6712 shared about 44.7% orthologue genes with its closest relative H. patelloides. Furthermore, 12 cryptic biosynthetic gene clusters (BGCs) with potential bioactivity, such as a mycosporine-like amino acid BGC, were detected. Interestingly, the full set of nitrogen fixation genes was found in H. disjuncta PCC 6712 despite its inability to grow on nitrogen-free medium. A comparison of genes responsible for multicellularity was performed, indicating that most of these genes were present and related to those found in other cyanobacterial orders. This is in contrast to the formation of pseudofilaments-a main feature of the genus Hyella-which is weakly expressed in H. disjuncta PCC 6712 but prominent in Hyella patelloides LEGE 07179. Thus, our study pinpoints crucial but hidden aspects of polyphasic cyanobacterial taxonomy.

Symphyonema bifilamentata sp. nov., the Right Fischerella ambigua 108b: Half a Decade of Research on Taxonomy and Bioactive Compounds in New Light.

Since 1965 a cyanobacterial strain termed 'Fischerella ambigua 108b' was the object of several studies investigating its potential as a resource for new bioactive compounds in several European institutes. Over decades these investigations uncovered several unique small molecules and their respective biosynthetic pathways, including the polychlorinated triphenyls of the ambigol family and the tjipanazoles. However, the true taxonomic character of the producing strain remained concealed until now. Applying a polyphasic approach considering the phylogenetic position based on the 16S rRNA and the protein coding gene rbcLX, secondary structures and morphological features, we present the strain 'Fischerella ambigua 108b' as Symphyonema bifilamentata sp. nov. 97.28. Although there is the type species (holotype) S. sinense C.-C. Jao 1944 there is no authentic living strain or material for genetic analyses for the genus Symphyonema available. Thus we suggest and provide an epitypification of S. bifilamentata sp. nov. 97.28 as a valid reference for the genus Symphyonema. Its affiliation to the family Symphyonemataceae sheds not only new light on this rare taxon but also on the classes of bioactive metabolites of these heterocytous and true-branching cyanobacteria which we report here. We show conclusively that the literature on the isolation of bioactive products from this organism provides further support for a clear distinction between the secondary metabolism of Symphyonema bifilamentata sp. nov. 97.28 compared to related and other taxa, pointing to the assignment of this organism into a separate genus.

Comparative proteomics of the toxigenic diazotroph Raphidiopsis raciborskii (cyanobacteria) in response to iron.

Raphidiopsis raciborskii is an invasive bloom-forming cyanobacteria with the flexibility to utilize atmospheric and fixed nitrogen. Since nitrogen-fixation has a high requirement for iron as an ezyme cofactor, we hypothesize that iron availability would determine the success of the species under nitrogen-fixing conditions. This study compares the proteomic response of cylindrospermopsin-producing and non-toxic strains of R. racibroskii to reduced iron concentrations, under nitrogen-fixing conditions, to examine any strain-specific adaptations that might increase fitness under these conditions. We also compared their proteomic responses at exponential and stationary growth phases to capture the changes throughout the growth cycle. Overall, the toxic strain was more competitive under Fe-starved conditions during exponential phase, with upregulated growth and transport-related proteins. The non-toxic strain showed reduced protein expression across multiple primary metabolism pathways. We propose that the increased expression of porin proteins during the exponential growth phase enables toxic strains to persist under Fe-starved conditions with this ability providing a potential explanation for the increased fitness of cylindrospermoipsin-producing strains during unfavourable environmental conditions.

Antarctic desert soil bacteria exhibit high novel natural product potential, evaluated through long-read genome sequencing and comparative genomics.

Actinobacteria and Proteobacteria are important producers of bioactive natural products (NP), and these phyla dominate in the arid soils of Antarctica, where metabolic adaptations influence survival under harsh conditions. Biosynthetic gene clusters (BGCs) which encode NPs, are typically long and repetitious high G + C regions difficult to sequence with short-read technologies. We sequenced 17 Antarctic soil bacteria from multi-genome libraries, employing the long-read PacBio platform, to optimize capture of BGCs and to facilitate a comprehensive analysis of their NP capacity. We report 13 complete bacterial genomes of high quality and contiguity, representing 10 different cold-adapted genera including novel species. Antarctic BGCs exhibited low similarity to known compound BGCs (av. 31%), with an abundance of terpene, non-ribosomal peptide and polyketide-encoding clusters. Comparative genome analysis was used to map BGC variation between closely related strains from geographically distant environments. Results showed the greatest biosynthetic differences to be in a psychrotolerant Streptomyces strain, as well as a rare Actinobacteria genus, Kribbella, while two other Streptomyces spp. were surprisingly similar to known genomes. Streptomyces and Kribbella BGCs were predicted to encode antitumour, antifungal, antibacterial and biosurfactant-like compounds, and the synthesis of NPs with antibacterial, antifungal and surfactant properties was confirmed through bioactivity assays.

Streptomyces sp. BV410 isolate from chamomile rhizosphere soil efficiently produces staurosporine with antifungal and antiangiogenic properties.

Applying a bioactivity-guided isolation approach, staurosporine was separated and identified as the active principle in the culture extract of the new isolate Streptomyces sp. BV410 collected from the chamomile rhizosphere. The biotechnological production of staurosporine by strain BV410 was optimized to yield 56 mg/L after 14 days of incubation in soy flour-glucose-starch-mannitol-based fermentation medium (JS). The addition of FeSO4 significantly improved the staurosporine yield by 30%, while the addition of ZnSO4 significantly reduced staurosporine yield by 62% in comparison with the starting conditions. Although staurosporine was first isolated in 1977 from Lentzea albida (now Streptomyces staurosporeus) and its potent kinase inhibitory effect has been established, here, the biological activity of this natural product was assessed in depth in vivo using a selection of transgenic zebrafish (Danio rerio) models, including Tg(fli1:EGFP) with green fluorescent protein-labeled endothelial cells allowing visualization and monitoring of blood vessels. This confirmed a remarkable antiangiogenic activity of the compound at doses of 1 ng/ml (2.14 nmol/L) which is below doses inducing toxic effects (45 ng/ml; 75 nmol/L). A new, efficient producing strain of commercially significant staurosporine has been described along with optimized fermentation conditions, which may lead to optimization of the staurosporine scaffold and its wider applicability.

Direct pathway cloning of the sodorifen biosynthetic gene cluster and recombinant generation of its product in E. coli.

BACKGROUND: Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene. RESULTS: Sequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA-D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible PtetO promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling. CONCLUSIONS: Genome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways.

Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles.

The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.

Physiological and Proteomic Responses of Continuous Cultures of Microcystis aeruginosa PCC 7806 to Changes in Iron Bioavailability and Growth Rate.

UNLABELLED: The hepatotoxin microcystin (MCYST) is produced by a variety of freshwater cyanobacterial species, including Microcystis aeruginosa Interestingly, MCYST-producing M. aeruginosa strains have been shown to outcompete their nontoxic counterparts under iron-limiting conditions. However, the reasons for this are unclear. Here we examined the proteomic response of M. aeruginosa PCC 7806 continuous cultures under different iron and growth regimes. Iron limitation was correlated with a global reduction in levels of proteins associated with energy metabolism and photosynthesis. These proteomic changes were consistent with physiological observations, including reduced chlorophyll a content and reduced cell size. While levels of MCYST biosynthesis proteins did not fluctuate during the study period, both intra- and extracellular toxin quotas were significantly higher under iron-limiting conditions. Our results support the hypothesis that intracellular MCYST plays a role in protecting the cell against oxidative stress. Further, we propose that extracellular MCYST may act as a signaling molecule, stimulating MCYST production under conditions of iron limitation and enhancing the fitness of bloom populations. IMPORTANCE: Microcystin production in water supply reservoirs is a global public health problem. Understanding the ecophysiology of hepatotoxic cyanobacteria, including their responses to the presence of key micronutrient metals such as iron, is central to managing harmful blooms. To our knowledge, this was the first study to examine proteomic and physiological changes occurring in M. aeruginosa continuous cultures under conditions of iron limitation at different growth rates.

Comparative proteomics reveals that a saxitoxin-producing and a nontoxic strain of Anabaena circinalis are two different ecotypes.

In Australia, saxitoxin production is restricted to the cyanobacterial species Anabaena circinalis and is strain-dependent. We aimed to characterize a saxitoxin-producing and nontoxic strain of A. circinalis at the proteomic level using iTRAQ. Seven proteins putatively involved in saxitoxin biosynthesis were identified within our iTRAQ experiment for the first time. The proteomic profile of the toxic A. circinalis was significantly different from the nontoxic strain, indicating that each is likely to inhabit a unique ecological niche. Under control growth conditions, the saxitoxin-producing A. circinalis displayed a higher abundance of photosynthetic, carbon fixation and nitrogen metabolic proteins. Differential abundance of these proteins suggests a higher intracellular C:N ratio and a higher concentration of intracellular 2-oxoglutarate in our toxic strain compared with the nontoxic strain. This may be a novel site for posttranslational regulation because saxitoxin biosynthesis putatively requires a 2-oxoglutarate-dependent dioxygenase. The nontoxic A. circinalis was more abundant in proteins, indicating cellular stress. Overall, our study has provided the first insight into fundamental differences between a toxic and nontoxic strain of A. circinalis, indicating that they are distinct ecotypes.

Brain dendritic cells: biology and pathology.

Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. In their quiescent and mature form, the presentation of self-antigens by DC leads to tolerance; whereas, antigen presentation by mature DC, after stimulation by pathogen-associated molecular patterns, leads to the onset of antigen-specific immunity. DC have been found in many of the major organs in mammals (e.g. skin, heart, lungs, intestines and spleen); while the brain has long been considered devoid of DC in the absence of neuroinflammation. Consequently, microglia, the resident immune cell of the brain, have been charged with many functional attributes commonly ascribed to DC. Recent evidence has challenged the notion that DC are either absent or minimal players in brain immune surveillance. This review will discuss the recent literature examining DC involvement within both the young and aged steady-state brain. We will also examine DC contributions during various forms of neuroinflammation resulting from neurodegenerative autoimmune disease, injury, and CNS infections. This review also touches upon DC trafficking between the central nervous system and peripheral immune compartments during viral infections, the new molecular technologies that could be employed to enhance our current understanding of brain DC ontogeny, and some potential therapeutic uses of DC within the CNS.

Viral-induced encephalitis initiates distinct and functional CD103+ CD11b+ brain dendritic cell populations within the olfactory bulb.

Dendritic cells (DC) are antigen-presenting cells found in both lymphoid and nonlymphoid organs, including the brain (bDC) of Cd11c/eyfp transgenic C57BL/6 mice. Using an intranasal vesicular stomatitis virus infection, we demonstrated that EYFP(+) cells amass in areas associated with viral antigens, take on an activated morphology, and project their processes into infected neuronal tissue within the olfactory bulb. These bDC separated into three EYFP(+) CD45(+) CD11b(+) populations, all but one being able to functionally promote both T lymphocyte proliferation and T(H)1 cytokine production. One population was shown to emanate from the brain and a second population was peripherally derived. The third population was of indeterminate origin, being both radiosensitive and not replenished by donor bone marrow. Finally, each EYFP(+) population contained CD11b(+) CD103(+) subpopulations and could be distinguished in terms of CD115, Gr-1, and Ly-6C expression, highlighting mucosal and monocyte-derived DC lineages.

A confocal and electron microscopic comparison of interferon beta-induced changes in vesicular stomatitis virus infection of neuroblastoma and nonneuronal cells.

Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. Pretreatment of sensitive cultured cells with IFNbeta results in a 10(4)-fold reduction in the release of infectious VSV particles. However, differences exist between the mechanisms of reduced infectious particle titers in cell lines of neuroblastoma and nonneuronal lineage. In L929-fibroblast-derived cells, using immunofluorescence confocal microscopy, infection under control conditions reveals the accumulation of VSV matrix, phosphoprotein (P), and nucleocapsid (N) proteins over time, with induced cellular morphological changes indicative of cytopathic effects (CPEs). Upon observing L929 cells that had been pretreated with IFNbeta, neither detectable VSV proteins nor CPEs were seen, consistent with type I IFN antiviral protection. When using the same techniques to observe VSV infections of NB41A3 cells, a neuroblastoma cell line, aside from similar viral progression in the untreated control cells, IFNbeta-treated cells illustrated a severely attenuated VSV infection. Attenuated VSV progression was observed through detection of VSV matrix, P, and N proteins in isolated cells during the first 8 h of infection. However, by 18-24 h postinfection all neuroblastomas had succumbed to the viral infection. Finally, upon closer inspection of IFNbeta-treated NB41A3 cells, no detectable changes in VSV protein localization were identified compared with untreated, virally infected neuroblastomas. Next, to extend our study to test our hypothesis that virion assembly is compromised within type I IFN-treated neuroblastoma cells, we employed electron microscopy to examine our experimental conditions at the ultrastructural level. Using VSV-specific antibodies in conjunction with immuno-gold reagents, we observed several similarities between the two cell lines, such as identification of viroplasmic regions containing VSV N and P proteins and signs of stress-induced CPEs of VSV-infected cells, which had either been mock-treated or pretreated with interferon-beta (IFNbeta). One difference we observed between nonneuronal and neuroblastoma cells was more numerous actively budding VSV virions across untreated L929 plasma membranes compared with untreated NB41A3 cells. Additionally, IFNbeta-treated, VSV-infected L929 cells exhibited neither cytoplasmic viroplasm nor viral protein expression. In contrast, IFNbeta-treated, VSV-infected NB41A3 cells showed evidence of VSV infection at a very low frequency as well as small-scale viroplasmic regions that colocalized with viral N and P proteins. Finally, we observed that VSV viral particles harvested from untreated VSV-infected L929 and NB41A3 cells were statistically similar in size and shape. A portion of VSV virions from IFNbeta-treated, virally infected NB41A3 cells were similar in size and shape to virus from both untreated cell types. However, among the sampling of virions, pleomorphic viral particles that were identified from IFNbeta-treated, VSV-infected NB41A3 cells were different enough to suggest a misassembly mechanism as part of the IFNbeta antiviral state in neuroblastoma cells.

IFN-beta-induced alteration of VSV protein phosphorylation in neuronal cells.

Vesicular stomatitis virus (VSV) replication is highly sensitive to interferon (IFN)-induced antiviral responses. VSV infection of well-known cell lines pretreated with IFN-beta results in a 10(4)-fold reduction in the release of infectious particles, with a concomitant abrogation in viral transcript and/or protein levels. However, in cell lines of neuronal lineage only a threefold reduction in viral transcript and protein levels was observed, despite the same 10(4)-fold reduction in released infectious virions, suggesting an assembly defect. Examination of VSV matrix (M) protein ubiquitination yielded no differences between mock- and IFN-beta-treated neuronal cells. Further analysis of potential post-translational modification events, by scintillation and two-dimensional electrophoretic methods, revealed IFN-beta-induced alterations in M protein and phosphoprotein (P) phosphorylation. Hypophosphorylated P protein was demonstrated by reduced (32)P counts, normalized by (35)S-cysteine/methionine incorporation, and by a shift in isoelectric focusing. Hypophosphorylation of VSV P protein was found to occur in neuronal cell lysates, but not within budded virions from the same IFN-beta-treated cells. In contrast, hyperphosphorylation of VSV M protein was observed in both cell lysates and viral particles from IFN-beta-treated neuronal cells. Hyperphosphorylated M protein was demonstrated by increased (32)P counts relative to (35)S-cysteine/methionine normalization, and by altered isoelectric focusing in protein populations from cell and viral lysates. Hyperphosphorylated VSV M protein was found to inhibit its association with VSV nucleocapsid, suggesting a possible mechanism for type I IFN-mediated misassembly through disruption of the interactions between ribonucleoprotein cores, and hyperphosphorylated M protein bound to the plasma membrane inner leaflet.

DISTINCT MECHANISMS OF INHIBITION OF VSV REPLICATION IN NEURONS MEDIATED BY TYPE I AND TYPE II IFN.

Acute viral infection of neurons presents a difficult problem to the host, since neurons are essential and not replaced, therefore cell-autonomous pathway(s) of suppressing viral replication are critical. We have examined the mechanisms by which neurons respond to exogenous interferons (IFNs) and observed that novel pathways inhibit acute vesicular stomatitis virus (VSV) replication. For both type I (IFN-beta) and Type II (IFN-gamma) interferons, post-translational modification of viral proteins contributed to the replication blockade, diminishing the efficiency of viral assembly and budding from the host neuron. IFN-gamma treatment induces the accumulation of NOS-1 in the absence of an increase of mRNA encoding this enzyme; a NOS-1-inhibiting protein, PIN, is rapidly ubiquitinated and eliminated in the presence of IFN-gamma. NOS-1 produces NO which combines with superoxide to form peroxynitrite (ONOO-), this binds tyrosines, cysteines, and serines; antagonism of NOS-1 with either non-specific or selective inhibitors block the antiviral effect of IFN-gamma. VSV proteins are decorated with -NO(2) in IFN-gamma-treated neurons, probably resulting in their diminished ability to interact properly and mature into budding virus. For IFN-beta, protein phosphorylation of the Matrix protein (M) and Phosphoprotein (P) were altered in infected neurons, with hyperphosphorylation of M (but not hypophosphorylated P) found in released virions. Hyperphosphorylated M protein does not immunoprecipitate with the viral ribonucleoprotein complex in IFN-beta-treated neurons. Thus both types of IFN interfere with viral assembly and release of infectious particles, but by distinct pathways.