Evolution-guided engineering of trans-acyltransferase polyketide synthases.

Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.

Draft genome sequences of 21 Pedobacter strains isolated from amphibian specimens.

The genomes of 21 Pedobacter strains isolated from the European salamander Salamandra salamandra and different Madagascan frog species were sequenced using Illumina sequencing. Here, we report their draft genome sequences (~4.7-7.2 Mbp in size) to allow comparative genomics and taxonomic assignment of these strains.

Characterization of an Orphan Type†III Polyketide Synthase Conserved in Uncultivated "Entotheonella" Sponge Symbionts.

Uncultivated bacterial symbionts from the candidate genus "Entotheonella" have been shown to produce diverse natural products previously attributed to their sponge hosts. In addition to these known compounds, "Entotheonella" genomes contain rich sets of biosynthetic gene clusters that lack identified natural products. Among these is a small type III polyketide synthase (PKS) cluster, one of only three clusters present in all known "Entotheonella" genomes. This conserved "Entotheonella" PKS (cep) cluster encodes the type III PKS CepA and the putative methyltransferase CepB. Herein, the characterization of CepA as an enzyme involved in phenolic lipid biosynthesis is reported. In vitro analysis showed a specificity for alkyl starter substrates and the production of tri- and tetraketide pyrones and tetraketide resorcinols. The conserved distribution of the cep cluster suggests an important role for the phenolic lipid polyketides produced in "Entotheonella" variants.

Amphibian skin-associated Pigmentiphaga: Genome sequence and occurrence across geography and hosts.

The bacterial communities colonizing amphibian skin have been intensively studied due to their interactions with pathogenic chytrid fungi that are causing drastic amphibian population declines. Bacteria of the family Alcaligenaceae, and more specifically of the genus Pigmentiphaga, have been found to be associated specifically to arboreal frogs. Here we analyze their occurrence in a previously assembled global skin microbiome dataset from 205 amphibian species. Pigmentiphaga made up about 5% of the total number of reads in this global dataset. They were mostly found in unrelated arboreal frogs from Madagascar (Mantellidae and Hyperoliidae), but also occurred at low abundances on Neotropical frogs. Based on their 16S sequences, most of the sequences belong to a clade within Pigmentiphaga not assignable to any type strains of the five described species of the genus. One isolate from Madagascar clustered with Pigmentiphaga aceris (>99% sequence similarity on 16S rRNA gene level). Here, we report the full genome sequence of this bacterium which, based on 16S sequences of >97% similarity, has previously been found on human skin, floral nectar, tree sap, stream sediment and soil. Its genome consists of a single circular chromosome with 6,165,255 bp, 5,300 predicted coding sequences, 57 tRNA genes, and three rRNA operons. In comparison with other known Pigmentiphaga genomes it encodes a higher number of genes associated with environmental information processing and cellular processes. Furthermore, it has a biosynthetic gene cluster for a nonribosomal peptide syntethase, and bacteriocin biosynthetic genes can be found, but clusters for ß-lactones present in other comparative Pigmentiphaga genomes are lacking.

Metabolic and evolutionary origin of actin-binding polyketides from diverse organisms.

Actin-targeting macrolides comprise a large, structurally diverse group of cytotoxins isolated from remarkably dissimilar micro- and macroorganisms. In spite of their disparate origins and structures, many of these compounds bind actin at the same site and exhibit structural relationships reminiscent of modular, combinatorial drug libraries. Here we investigate biosynthesis and evolution of three compound groups: misakinolides, scytophycin-type compounds and luminaolides. For misakinolides from the sponge Theonella swinhoei WA, our data suggest production by an uncultivated 'Entotheonella' symbiont, further supporting the relevance of these bacteria as sources of bioactive polyketides and peptides in sponges. Insights into misakinolide biosynthesis permitted targeted genome mining for other members, providing a cyanobacterial luminaolide producer as the first cultivated source for this dimeric compound family. The data indicate that this polyketide family is bacteria-derived and that the unusual macrolide diversity is the result of combinatorial pathway modularity for some compounds and of convergent evolution for others.

Injury-induced immune responses in Hydra.

The impact of injury-induced immune responses on animal regenerative processes is highly variable, positive or negative depending on the context. This likely reflects the complexity of the innate immune system that behaves as a sentinel in the transition from injury to regeneration. Early-branching invertebrates with high regenerative potential as Hydra provide a unique framework to dissect how injury-induced immune responses impact regeneration. A series of early cellular events likely require an efficient immune response after amputation, as antimicrobial defence, epithelial cell stretching for wound closure, migration of interstitial progenitors toward the wound, cell death, phagocytosis of cell debris, or reconstruction of the extracellular matrix. The analysis of the injury-induced transcriptomic modulations of 2636 genes annotated as immune genes in Hydra identified 43 genes showing an immediate/early pulse regulation in all regenerative contexts examined. These regulations point to an enhanced cytoprotection via ROS signaling (Nrf, C/EBP, p62/SQSMT1-l2), TNFR and TLR signaling (TNFR16-like, TRAF2l, TRAF5l, jun, fos-related, SIK2, ATF1/CREB, LRRC28, LRRC40, LRRK2), proteasomal activity (p62/SQSMT1-l1, Ced6/Gulf, NEDD8-conjugating enzyme Ubc12), stress proteins (CRYAB1, CRYAB2, HSP16.2, DnaJB9, HSP90a1), all potentially regulating NF-κB activity. Other genes encoding immune-annotated proteins such as NPYR4, GTPases, Swap70, the antiproliferative BTG1, enzymes involved in lipid metabolism (5-lipoxygenase, ACSF4), secreted clotting factors, secreted peptidases are also pulse regulated upon bisection. By contrast, metalloproteinases and antimicrobial peptide genes largely follow a context-dependent regulation, whereas the protease inhibitor α2macroglobulin gene exhibits a sustained up-regulation. Hence a complex immune response to injury is linked to wound healing and regeneration in Hydra.

Recent advances in genome-based polyketide discovery.

Polyketides are extraordinarily diverse secondary metabolites of great pharmacological value and with interesting ecological functions. The post-genomics era has led to fundamental changes in natural product research by inverting the workflow of secondary metabolite discovery. As opposed to traditional bioactivity-guided screenings, genome mining is an in silico method to screen and analyze sequenced genomes for natural product biosynthetic gene clusters. Since genes for known compounds can be recognized at the early computational stage, genome mining presents an opportunity for dereplication. This review highlights recent progress in bioinformatics, pathway engineering and chemical analytics to extract the biosynthetic secrets hidden in the genome of both well-known natural product sources as well as previously neglected bacteria.

Cell death: a program to regenerate.

Recent studies in Drosophila, Hydra, planarians, zebrafish, mice, indicate that cell death can open paths to regeneration in adult animals. Indeed injury can induce cell death, itself triggering regeneration following an immediate instructive mechanism, whereby the dying cells release signals that induce cellular responses over short and/or long-range distances. Cell death can also provoke a sustained derepressing response through the elimination of cells that suppress regeneration in homeostatic conditions. Whether common properties support what we name "regenerative cell death," is currently unclear. As key parameters, we review here the injury proapoptotic signals, the signals released by the dying cells, the cellular responses, and their respective timing. ROS appears as a common signal triggering cell death through MAPK and/or JNK pathway activation. But the modes of ROS production vary, from a brief pulse upon wounding, to repeated waves as observed in the zebrafish fin where ROS supports two peaks of cell death. Indeed regenerative cell death can be restricted to the injury phase, as in Hydra, Drosophila, or biphasic, immediate, and delayed, as in planarians and zebrafish. The dying cells release in a caspase-dependent manner a variety of signaling molecules, cytokines, growth factors, but also prostaglandins or ATP as recorded in Drosophila, Hydra, mice, and zebrafish, respectively. Interestingly, the ROS-producing cells often resist to cell death, implying a complex paracrine mode of signaling to launch regeneration, involving ROS-producing cells, ROS-sensing cells that release signaling molecules upon caspase activation, and effector cells that respond to these signals by proliferating, migrating, and/or differentiating.

Hydra, a versatile model to study the homeostatic and developmental functions of cell death.

In the freshwater cnidarian polyp Hydra, cell death takes place in multiple contexts. Indeed apoptosis occurs during oogenesis and spermatogenesis, during starvation, and in early head regenerating tips, promoting local compensatory proliferation at the boundary between heterografts. Apoptosis can also be induced upon exposure to pro-apoptotic agents (colchicine, wortmannin), upon heat-shock in the thermosensitive sf-1 mutant, and upon wounding. In all these contexts, the cells that undergo cell death belong predominantly to the interstitial cell lineage, whereas the epithelial cells, which are rather resistant to pro-apoptotic signals, engulf the apoptotic bodies. Beside this clear difference between the interstitial and the epithelial cell lineages, the different interstitial cell derivatives also show noticeable variations in their respective apoptotic sensitivity, with the precursor cells appearing as the most sensitive to pro-apoptotic signals. The apoptotic machinery has been well conserved across evolution. However, its specific role and regulation in each context are not known yet. Tools that help characterize apoptotic activity in Hydra have recently been developed. Among them, the aposensor Apoliner initially designed in Drosophila reliably measures wortmannin-induced apoptotic activity in a biochemical assay. Also, flow cytometry and TUNEL analyses help identify distinctive features between wortmannin-induced and heat-shock induced apoptosis in the sf-1 strain. Thanks to the live imaging tools already available, Hydra now offers a model system with which the functions of the apoptotic machinery to maintain long-term homeostasis, stem cell renewal, germ cell production, active developmental processes and non-self response can be deciphered.

Bit-1 mediates integrin-dependent cell survival through activation of the NFkappaB pathway.

Loss of properly regulated cell death and cell survival pathways can contribute to the development of cancer and cancer metastasis. Cell survival signals are modulated by many different receptors, including integrins. Bit-1 is an effector of anoikis (cell death due to loss of attachment) in suspended cells. The anoikis function of Bit-1 can be counteracted by integrin-mediated cell attachment. Here, we explored integrin regulation of Bit-1 in adherent cells. We show that knockdown of endogenous Bit-1 in adherent cells decreased cell survival and re-expression of Bit-1 abrogated this effect. Furthermore, reduction of Bit-1 promoted both staurosporine and serum-deprivation induced apoptosis. Indeed knockdown of Bit-1 in these cells led to increased apoptosis as determined by caspase-3 activation and positive TUNEL staining. Bit-1 expression protected cells from apoptosis by increasing phospho-IκB levels and subsequently bcl-2 gene transcription. Protection from apoptosis under serum-free conditions correlated with bcl-2 transcription and Bcl-2 protein expression. Finally, Bit-1-mediated regulation of bcl-2 was dependent on focal adhesion kinase, PI3K, and AKT. Thus, we have elucidated an integrin-controlled pathway in which Bit-1 is, in part, responsible for the survival effects of cell-ECM interactions.

Rapid identification and semi-quantitative determination of polymer additives by desorption electrospray ionization/time-of-flight mass spectrometry.

A fast and simple method for the direct qualitative and semi-quantitative determination of a set of four polymer additives in plastic samples by desorption electrospray ionization time-of-flight mass spectrometry (DESI-TOF-MS) is presented. After evaluation of crucial DESI parameters such as composition of spray solutions and spray voltages, a series of lab-made polypropylene samples containing Chimassorb 81 (2-hydroxy-4-n-octoxybenzophenone), Tinuvin 328 (2-(2-hydroxy-3, 5-ditert-pentylphenyl)-benzotriazole), Tinuvin 326 (2-(2-hydroxy-3-tert-butyl-5-methylphenyl)-5-chloro benzotriazole), and Tinuvin 770 (bis(2,2,6,6,-tetramethyl-4-piperidyl)sebaceate) in concentrations between 0.02% and 0.2% were analyzed, resulting in calibration graphs with R (2) better than 0.994. To demonstrate the applicability of the developed method for the investigation of real samples, liners for in-ground swimming pools and polypropylene granules were analyzed with respect to their content in the selected polymer additives. Two alternative methods, both well established in the fields of polymer additive analysis, namely HPLC with UV detection (after previous extraction) and thermodesorption gas chromatography/mass spectrometry have been employed for evaluation of the results from the DESI experiments.