Structure and folding of four putative kink turns identified in structured RNA species in a test of structural prediction rules.

k-Turns are widespread key architectural elements that occur in many classes of RNA molecules. We have shown previously that their folding properties (whether or not they fold into their tightly kinked structure on addition of metal ions) and conformation depend on their local sequence, and we have elucidated a series of rules for prediction of these properties from sequence. In this work, we have expanded the rules for prediction of folding properties, and then applied the full set to predict the folding and conformation of four probable k-turns we have identified amongst 224 structured RNA species found in bacterial intergenenic regions by the Breaker lab (1). We have analyzed the ion-dependence of folding of the four k-turns using fluorescence resonance energy transfer, and determined the conformation of two of them using X-ray crystallography. We find that the experimental data fully conform to both the predicted folding and conformational properties. We conclude that our folding rules are robust, and can be applied to new k-turns of unknown characteristics with confidence.

Cell-to-cell interaction requires optimal positioning of a pilus tip adhesin modulated by gram-positive transpeptidase enzymes.

Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.

Dereplication of microbial metabolites through database search of mass spectra.

Natural products have traditionally been rich sources for drug discovery. In order to clear the road toward the discovery of unknown natural products, biologists need dereplication strategies that identify known ones. Here we report DEREPLICATOR+, an algorithm that improves on the previous approaches for identifying peptidic natural products, and extends them for identification of polyketides, terpenes, benzenoids, alkaloids, flavonoids, and other classes of natural products. We show that DEREPLICATOR+ can search all spectra in the recently launched Global Natural Products Social molecular network and identify an order of magnitude more natural products than previous dereplication efforts. We further demonstrate that DEREPLICATOR+ enables cross-validation of genome-mining and peptidogenomics/glycogenomics results.

Screening and purification of natural products from actinomycetes that affect the cell shape of fission yeast.

This study was designed to identify bioactive compounds that alter the cellular shape of the fission yeast Schizosaccharomyces pombe by affecting functions involved in the cell cycle or cell morphogenesis. We used a multidrug-sensitive fission yeast strain, SAK950 to screen a library of 657 actinomycete bacteria and identified 242 strains that induced eight different major shape phenotypes in S. pombe These include the typical cell cycle-related phenotype of elongated cells, and the cell morphology-related phenotype of rounded cells. As a proof of principle, we purified four of these activities, one of which is a novel compound and three that are previously known compounds, leptomycin B, streptonigrin and cycloheximide. In this study, we have also shown novel effects for two of these compounds, leptomycin B and cycloheximide. The identification of these four compounds and the explanation of the S. pombe phenotypes in terms of their known, or predicted bioactivities, confirm the effectiveness of this approach.

Progressive Stereo Locking (PSL): A Residual Dipolar Coupling Based Force Field Method for Determining the Relative Configuration of Natural Products and Other Small Molecules.

Establishing the relative configuration of a bioactive natural product represents the most challenging part in determining its structure. Residual dipolar couplings (RDCs) are sensitive probes of the relative spatial orientation of internuclear vectors. We adapted a force field structure calculation methodology to allow free sampling of both R and S configurations of the stereocenters of interest. The algorithm uses a floating alignment tensor in a simulated annealing protocol to identify the conformations and configurations that best fit experimental RDC and distance restraints (from NOE and J-coupling data). A unique configuration (for rigid molecules) or a very small number of configurations (for less rigid molecules) of the structural models having the lowest chiral angle energies and reasonable magnitudes of the alignment tensor are provided as the best predictions of the unknown configuration. For highly flexible molecules, the progressive locking of their stereocenters into their statistically dominant R or S state dramatically reduces the number of possible relative configurations. The result is verified by checking that the same configuration is obtained by initiating the locking from different regions of the molecule. For all molecules tested having known configurations (with conformations ranging from mostly rigid to highly flexible), the method accurately determined the correct configuration.

Evaluation of the bioactive extract of actinomyces isolated from the Egyptian environment against aflatoxin B1-induce cytotoxicity, genotoxicity and oxidative stress in the liver of rats.

This study aimed to determine the bioactive compounds of actinomyces (ACT) isolated from the Egyptian environment (D-EGY) and to evaluate their protective activity against AFB1 in female Sprague-Dawley rats. Six groups of animals were treated orally for 3 weeks included: C, the control group, T1, AFB1-treated group (80 µg/kg b.w), T2 and T3, the groups received ACT extract at low (25 mg/kg b.w) or high (50 mg/kg b.w) doses, T4 and T5, the groups received AFB1 plus the low or high dose of ACT extract. Blood, bone marrow and tissue samples were collected for different analyses and histological examination. The results revealed the identification of 40 components, representing 99.98%. Treatment with AFB1 disturbs liver function parameters, oxidative stress markers, antioxidant gene expressions, DNA fragmentation and induced severe histological changes. ACT extract at the low or high doses did not induce significant changes in all the tested parameters or histological picture of the liver. Moreover, ACT extract succeeded to induce a significant protection against the toxicity of AFB1. It could be concluded that the bioactive compounds in ACT are promise candidate for the development of food additive or drugs for the protection and treatment of liver disorders in the endemic area.

[Screening, identification and antimicrobial activity of alkaloid produced by endophytic actinomycetes from Fritillaria unibracteata in western Sichuan plateau].

To explore the resource of endophytic actinomycete in Fritillaria unibracteata, and alleviate the shortage of F. unibracteata resource, using F. unibracteata as experimental materials which growth in the western Sichuan plateau and cut its healthy bulb. Pure culture, insert, TLC and Oxford cup were applied to observe the mycelial morphology, research the ability of producing alkaloid and its antibacterial activity. Totally, 14 endophytic actinomycete strains were isolated by using Gao culture media. Based on the color reaction, 5 typical strains were selected for producing alkaloid. Through the TLC technique, all strains produced 2 obvious alkaloids spots. Antibacterial activity determination showed that the antimicrobial effects of 2 strains is prominent, the diameter up to 11 mm.16S rRNA gene sequence comparison analysis showed that 5 strains belonging to the Streptomyces. The alkaloids produced by endophytic actinomycetes are not related to F. unibracteata, but its fermentation liquid has antibacterial effect, it is worthy of further study.

Antimycin-type depsipeptides: discovery, biosynthesis, chemical synthesis, and bioactivities.

Covering: up to 2016Antimycin-type depsipeptides are a family of natural products with great structural diversity and outstanding biological activities. These compounds have typically been isolated from actinomycetes and are generated from hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly lines. This review covers the literature on the four classes of antimycin-type depsipeptides, which differ by macrolactone ring size, and it discusses the discovery, biosynthesis, chemical synthesis, and biological activities of this family of compounds.

Engineering microbial hosts for production of bacterial natural products.

Covering up to end 2015Microbial fermentation provides an attractive alternative to chemical synthesis for the production of structurally complex natural products. In most cases, however, production titers are low and need to be improved for compound characterization and/or commercial production. Owing to advances in functional genomics and genetic engineering technologies, microbial hosts can be engineered to overproduce a desired natural product, greatly accelerating the traditionally time-consuming strain improvement process. This review covers recent developments and challenges in the engineering of native and heterologous microbial hosts for the production of bacterial natural products, focusing on the genetic tools and strategies for strain improvement. Special emphasis is placed on bioactive secondary metabolites from actinomycetes. The considerations for the choice of host systems will also be discussed in this review.

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks.

Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker intermediates that recover strength with time or ligand binding. Based on the high force extensibility, CnaA-containing pili can dissipate ∼28-fold as much energy compared with their inextensible counterparts before reaching forces sufficient to cleave covalent bonds. We propose that efficient mechanical energy dissipation is key for sustained bacterial attachment against mechanical perturbations.

Prevalence of Actinomyces spp. in patients with chronic periodontitis.

This study investigated the prevalence of Actinomyces spp. in shallow, deep and very deep pockets of patients with chronic periodontitis compared to healthy controls and correlated the results with clinical status. Twenty patients with chronic periodontitis and 15 healthy subjects were enrolled in this study. Clinical indices were recorded in a six-point measurement per tooth. From each patient samples of supra and subgingival plaque were taken separately from teeth with shallow, deep and very deep pockets. Samples of supragingival plaque and sulcular microflora were collected from the healthy subjects. All the samples were cultivated on different media at 37̊C in an anaerobic atmosphere for 7 days. All the suspect colonies were identified using a rapid ID 32 A system (bioMèrieux) and MALDI-TOF-MS analysis using an Autoflex II Instrument (Bruker Daltonics) together with in house developed identification software and a reference spectra database. A total of 977 strains were identified as Actinomyces. Actinomyces naeslundii/oris/johnsonii (430 isolates) was the most prevalent species and was found in all patients and in almost all of the healthy subjects. Significant differences (p=0.003) between the groups were found for Actinomyces odontolyticus/meyeri and Actinomyces israelii which were associated with periodontitis patients. Actinomyces dentalis was found in higher percentage (p=0.015) in the periodontitis group. Actinomyces gerencseriae and Actinomyces massiliensis were significantly more often found supragingivally than subgingivally (p=0.004, p=0.022, respectively) in the periodontitis group. Whether some Actinomyces species, definitely important plaque formers, are actively involved in the pathogenicity of chronic periodontitis needs further investigation.

A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria.

Marine Actinomycetes as potential source for histone deacetylase inhibitors and epigenetic modulation.

UNLABELLED: In the light of important detrimental role of aberrant histone deacetylases (HDAC) production during various clinical complications, development of therapeutically effective and specific inhibitors of HDAC is critically important. This study deals with the screening for HDAC inhibitors from marine Actinomycetes. The isolation of Actinomycetes from 22 sediment samples along the Southern Coast of India yielded 186 strains including Streptomyces, Nocardipsis, evaluated for HDAC inhibition using HeLa cells. Among the 186 isolates, 10 strains have shown moderate to strong inhibition. The maximum inhibition (61%) was seen with strain VITKSM06 and least inhibition (31%) was seen with strain VITSJT03. The MTT cell proliferation assay using HeLa cell line showed significant cytotoxicity with an IC50 of 5·9 µg ml(-1) by VITKSM06-derived metabolite and 26·2 µg ml(-1) by VITSJT03. The compound treated HeLa cells displayed an altered morphology and condensed chromatin which may be due to HDAC inhibition. Based on the phylogenetic analysis, the potential strains were identified as Nocardiopsis sp VITKSM06, Streptomyces sp VITAKS1 and Streptomyces sp VITRSM02. This study reveals the importance of screening marine Actinomycetes for the discovery of potential novel HDAC inhibitors of therapeutic importance. SIGNIFICANCE AND IMPACT OF THE STUDY: Histone deacetylases (HDAC) are epigenetic enzymes that regulate the deacetylation in lysine group on a histone, and thus regulate the gene expression. The HDAC inhibitors are reported to promote apoptosis on tumour cells, thus become clinically important drug target. Several studies have addressed the identification of putative HDAC inhibitors as therapeutic agents for cancer and until now those cleared phase III human trials are very limited. This study attempts to investigate the chemical diversity found in marine Actinomycetes towards negative HDAC modulation, which could be used individually or in combination as anti-cancerous and other therapeutic measure.

Acetylcholinesterase inhibitory dimeric indole derivatives from the marine actinomycetes Rubrobacter radiotolerans.

Investigation of the bioactive secondary metabolites of the marine actinomycetes Rubrobacter radiotolerans led to the isolation and characterization of two naturally rare dimeric indole derivatives (1 and 2). The structures of these new compounds were elucidated by spectroscopic data interpretation, and the absolute configurations were assigned by CD calculations. The acetylcholinesterase (AchE) inhibitory activity of compounds 1 and 2 was evaluated, both of which showed moderate activity with IC50 values of 11.8 and 13.5µM, respectively.

An efficient synthesis method targeted to marine alkaloids marinacarbolines A-D and their antitumor activities.

Marinacarbolines A-D are a series of marine ß-carboline alkaloids isolated from actinomycete Marinactinospora thermotolerans of the deep South China Sea with antiplasmodial activities. In inhibition assays of in vitro growth of Plasmodium falciparum, marinacarbolines exhibited antiplasmodial activity against drug-sensitive line 3D7 and drug-resistant line Dd2 of P. falciparum. However, approaches for the synthesis of such useful compounds are very limited. In this work, we reported a simple, efficient, and versatile process to synthesize marinacarbolines A-D (1-4). On the basis of that, the antitumor activities of marinacarbolines in a structure-dependent manner were allowed to be unveiled.

Isolation and structure elucidation of cystargamide, a lipopeptide from Kitasatospora cystarginea.

A new lipopeptide, cystargamide (1) was isolated from the fermentation broth of the actinomycete Kitasatospora cystarginea. The bacterial strain was selected from a set of 12 Kitasatospora spp. using a secondary metabolomics approach combining liquid chromatography/high-resolution mass spectrometry (LC-HRMS) with principal component analysis (PCA). Cystargamide (1) was purified by reversed-phase HPLC, and the structure elucidation was achieved by interpreting mass spectrometry and NMR data. Cystargamide (1) contains rare structural features including a 5-hydroxy tryptophan residue and a 2,3-epoxydecanoyl fatty acid group.

Imaging mass spectrometry reveals highly specific interactions between actinomycetes to activate specialized metabolic gene clusters.

The genomes of actinomycetes contain numerous gene clusters potentially able to encode the production of many antibiotics and other specialized metabolites that are not expressed during growth under typical laboratory conditions. Undoubtedly, this reflects the soil habitat of these organisms, which is highly complex physically, chemically, and biotically; the majority of the compounds that make up the specialized metabolome are therefore adaptive only under specific conditions. While there have been numerous previous reports of "waking up" the "sleeping" gene clusters, many involving genetic interventions or nutritional challenges, the role of competing microorganisms has been comparatively little studied. Now, Traxler et al. [M. F. Traxler, J. D. Watrous, T. Alexandrov, P. C. Dorrestein, and R. Kolter, mBio 4(4):e00459-13, 2013, doi:10.1128/mBio.00459-13] have used the recently described technique of microscale imaging mass spectrometry to analyze in detail the stimulation of specialized metabolite production by the model actinomycete Streptomyces coelicolor A3(2) by growth in proximity to other actinomycetes. The striking finding from these experiments was that growth of S. coelicolor close to each of the five other actinomycetes studied caused it to produce many specialized metabolites that were not made when it was grown in isolation and that the majority of the compounds were interaction specific, i.e., they occurred only in one of the five pairwise combinations, emphasizing the highly specific nature of the interactions. These observations contribute substantially to the increasing awareness of communication between microorganisms in complex natural communities, as well as auguring well for the discovery of useful specialized metabolites based on microbial interactions.

Identification of a novel 16S rRNA gene variant of Actinomyces funkei from six patients with purulent infections.

Little is known about the clinical significance and laboratory diagnosis of Actinomyces funkei. In this report we describe six clinical cases where A. funkei was isolated from purulent, polymicrobial infections. Conventional identification procedures were compared with molecular methods including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique. Analysis of the full 16S rRNA gene sequence of the six investigated strains revealed differences from the A. funkei type strain. DNA-DNA hybridization showed that the clinical strains represent a novel 16S rRNA gene variant within the species of A. funkei.

Marine natural products.

This review covers the literature published in 2011 for marine natural products, with 870 citations (558 for the period January to December 2011) referring to compounds isolated from marine microorganisms and phytoplankton, green, brown and red algae, sponges, cnidarians, bryozoans, molluscs, tunicates, echinoderms, mangroves and other intertidal plants and microorganisms. The emphasis is on new compounds (1152 for 2011), together with the relevant biological activities, source organisms and country of origin. Biosynthetic studies, first syntheses, and syntheses that lead to the revision of structures or stereochemistries, have been included.

[Screening of antifungi endophytic actinomyces strains from salvia przewalskii in Tibean Plateau].

Twenty-four endophytic actinomycetes strains were isolated from the Salvia przewalskii in Tibetan Plateau of China by tablet coating method. Fusarium moniliforme, Helminthosporium turcicum and Bipolaris maydis were selected as indicator fungi to test the antimicrobial activities of these endophytic actinomycetes by tablet confrontation method. The results showed that 21 strains can produce antimicrobial substances which accounts for 85.7% of the total separates number. Four strains of endogenous actinomyces have more obvious antifungi activity. According to results of morphology and culture properties and 16S rDNA sequences of endophytic actinomyces, it is concluded that all of the isolates were streptomycetes trains.