Diversity and Functional Evolution of Terpene Synthases in Dictyostelid Social Amoebae

Dictyostelids, or social amoebae, have a unique life style in forming multicellular fruiting bodies from unicellular amoeboids upon starvation. Recently, dictyostelids were found to contain terpene synthase (TPS) genes, a gene type of secondary metabolism previously known to occur only in plants, fungi and bacteria. Here we report an evolutionary functional study of dictyostelid TPS genes. The number of TPS genes in six species of dictyostelids examined ranges from 1 to 19; and the model species Dictyostelium purpureum contains 12 genes. Using in vitro enzyme assays, the 12 TPS genes from D. purpureum were shown to encode functional enzymes with distinct product profiles. The expression of the 12 TPS genes in D. purpureum is developmentally regulated. During multicellular development, D. purpureum releases a mixture of volatile terpenes dominated by sesquiterpenes that are the in vitro products of a subset of the 12 TPS genes. The quality and quantity of the terpenes released from D. purpureum, however, bear little resemblance to those of D. discoideum, a closely related dictyostelid. Despite these variations, the conserved clade of dictyostelid TPSs, which have an evolutionary distance of more than 600 million years, has the same biochemical function, catalyzing the formation of a sesquiterpene protoillud-7-ene. Taken together, our results indicate that the dynamic evolution of dictyostelid TPS genes includes both purifying selection of an orthologous group and species-specific expansion with functional divergence. Consequently, the terpenes produced by these TPSs most likely have conserved as well as speciesadaptive biological functions as chemical languages in dictyostelids

Dissecting the functional role of polyketide synthases in Dictyostelium discoideum

Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds

Two functionally distinctive phosphopantetheinyl transferases from amoeba Dictyostelium discoideum

The life cycle of Dictyostelium discoideum is proposed to be regulated by expression of small metabolites. Genome sequencing studies have revealed a remarkable array of genes homologous to polyketide synthases (PKSs) that are known to synthesize secondary metabolites in bacteria and fungi. A crucial step in functional activation of PKSs involves their post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases). PPTases have been recently characterized from several bacteria; however, their relevance in complex life cycle of protozoa remains largely unexplored. Here we have identified and characterized two phosphopantetheinyl transferases from D. discoideum that exhibit distinct functional specificity. DiAcpS specifically modifies a stand-alone acyl carrier protein (ACP) that possesses a mitochondrial import signal. DiSfp in contrast is specific to Type I multifunctional PKS/fatty acid synthase proteins and cannot modify the stand-alone ACP. The mRNA of two PPTases can be detected during the vegetative as well as starvation-induced developmental pathway and the disruption of either of these genes results in non-viable amoebae. Our studies show that both PPTases play an important role in Dictyostelium biology and provide insight into the importance of PPTases in lower eukaryotes

Comparative genome and transcriptome analyses of the social amoeba Acytostelium subglobosum that accomplishes multicellular development without germ-soma differentiation

Background Social amoebae are lower eukaryotes that inhabit the soil. They are characterized by the construction of a starvation-induced multicellular fruiting body with a spore ball and supportive stalk. In most species, the stalk is filled with motile stalk cells, as represented by the model organism Dictyostelium discoideum, whose developmental mechanisms have been well characterized. However, in the genus Acytostelium, the stalk is acellular and all aggregated cells become spores. Phylogenetic analyses have shown that it is not an ancestral genus but has lost the ability to undergo cell differentiation. Results We performed genome and transcriptome analyses of Acytostelium subglobosum and compared our findings to other available dictyostelid genome data. Although A. subglobosum adopts a qualitatively different developmental program from other dictyostelids, its gene repertoire was largely conserved. Yet, families of polyketide synthase and extracellular matrix proteins have not expanded and a serine protease and ABC transporter B family gene, tagA, and a few other developmental genes are missing in the A. subglobosum lineage. Temporal gene expression patterns are astonishingly dissimilar from those of D. discoideum, and only a limited fraction of the ortholog pairs shared the same expression patterns, so that some signaling cascades for development seem to be disabled in A. subglobosum. Conclusions The absence of the ability to undergo cell differentiation in Acytostelium is accompanied by a small change in coding potential and extensive alterations in gene expression patterns

Mining microbial genomes for new natural products and biosynthetic pathways

Analyses of microbial genome sequences have revealed numerous examples of ‘cryptic’ or ‘orphan’ biosynthetic gene clusters, with the potential to direct the production of novel, structurally complex natural products. This article summarizes the various methods that have been developed for discovering the products of cryptic biosynthetic gene clusters in microbes and gives an account of my group's discovery of the products of two such gene clusters in the model actinomycete Streptomyces coelicolor M145. These discoveries hint at new mechanisms, roles and specificities for natural product biosynthetic enzymes. Our efforts to elucidate these are described. The identification of new secondary metabolites of S. coelicolor raises the question: what is their biological function? Progress towards answering this question is also summarized

Biosynthesis and Roles of Virulence Conferring Cell Wall Associated Dimycocerosate Esters in \u3cem\u3eMycobacterium marinum\u3c/em\u3e

Mycobacterial species include a variety of obligate and opportunistic pathogens that cause several important diseases affecting mankind such as tuberculosis and leprosy. The most unique feature of these bacteria is their intricate cell wall that poses a permeability barrier to antibiotics and contributes to their pathogenicity and persistence within the host. The cell wall hosts several complex lipids such as dimycocerosate esters (DIMs), which are found in many clinically relevant pathogenic species of mycobacteria. DIMs have been implicated in the virulence of mycobacteria and play a major role in helping the bacteria evade host immune responses. It is therefore crucial to define the biosynthesis and role of DIMs in mycobacteria, to better understand these organisms and identify new drug target candidates. DIMs consist of two structurally related groups: phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs). PDIMs and PGLs share part of a biosynthetic pathway that consists of two enzyme families: polyketide synthases (PKSs) and fatty acyl AMP ligases (FAALs). This dissertation has investigated the roles of PKSs and FAALs during PGL biosynthesis in the pathogenic nontuberculous mycobacterium; Mycobacterium marinum. More specifically, it is focused on mutational studies that probed the mechanism by which intermediates synthesized by an iterative PKS, Pks15/1 are transferred to a non-iterative PKS, PpsA during PGL biosynthesis. Our findings specified the role of the loading acyl carrier protein domain of PpsA, in the capture of intermediates from Pks15/1 during PGL biosynthesis. We also provided the first evidence supporting a model in which the transfer of intermediates during PGL biosynthesis is dependent on a novel FAAL enzyme (FadD29) that acts as an intermediary between Pks15/1 and PpsA, within a nontuberculous mycobacterial species. This dissertation has also explored the hypothesis that different gene knockouts that render the same PDIM and/or PGL deficiency phenotypes lead to strains with equivalent pleiotropic profiles. The availability of six M. marinum mutants, each with a different gene knockout in the PDIM/PGL biosynthetic pathway, provided an opportunity to probe for the pleiotropic consequences of gene knockouts leading to PDIMˉ PGLˉ, PDIM+ PGLˉ, or PDIMˉ PGL+ phenotypes. We evaluated the mutants for changes in cell surface properties, cell envelope permeability, antimicrobial drug susceptibility, biofilm formation virulence in an amoeba model system, sliding motility and in vitro growth assays. Our results revealed that the pleiotropic patterns emerging from the different gene knockouts lead to: altered cell surface properties, weakened cell envelope permeability barrier, increased antibiotic susceptibility, reduced biofilm formation and different attenuation levels in an amoeba model. No notable differences were observed in sliding motility and in vitro growth of the different mutants. Our findings also advocate that, different enzymes of the pathway whose elimination equally leads to PDIM and PGL deficiency might not be equivalent drug target candidates

Od sekvencije DNA do kemijske strukture – pretraživanje mikrobnih genomskih i metagenomskih skupova podataka radi pronalaženja novih prirodnih spojeva

Rapid mining of large genomic and metagenomic data sets for modular polyketide synthases, non-ribosomal peptide synthetases and hybrid polyketide synthase/non-ribosomal peptide synthetase biosynthetic gene clusters has been achieved using the generic computer program packages ClustScan and CompGen. These program packages perform the annotation with the hierarchical structuring into polypeptides, modules and domains, as well as storage and graphical presentations of the data. This aims to achieve the most accurate predictions of the activities and specificities of catalytically active domains that can be made with present knowledge, leading to a prediction of the most likely chemical structures produced by these enzymes. The program packages also allow generation of novel clusters by homologous recombination of the annotated genes in silico. ClustScan and CompGen were used to construct a custom database of known compounds (CSDB) and of predicted entirely novel recombinant products (r-CSDB) that can be used for in silico screening with computer aided drug design technology. The use of these programs has been exemplified by analysing genomic sequences from terrestrial prokaryotes and eukaryotic microorganisms, a marine metagenomic data set and a newly discovered example of a \u27shared metabolic pathway\u27 in marine-microbial endosymbiosis.Brzo pretraživanje genomskih i metagenomskih skupova podataka, modularnih biosintetskih genskih nakupina poliketid sintaza i sintetaza neribosomalno sintetiziranih peptida, postignuto je primjenom generičkih računalnih programskih paketa ClustScan i CompGen. Ti programski paketi provode anotaciju hijerarhijskim strukturiranjem podataka na polipeptide, module i domene, te pohranu i grafičku prezentaciju tih podataka. Na temelju dosadašnjih spoznaja, nastoji se postići najtočnije moguće predviđanje aktivnosti i specifičnosti katalitički aktivnih domena, što vodi prema predviđanju najvjerojatnijih kemijskih struktura koje ti enzimi mogu sintetizirati. Programski paketi ClustScan i CompGen omogućuju generiranje novih genskih nakupina homolognom rekombinacijom anotiranih gena u uvjetima in silico, a upotrijebljeni su i za konstrukciju vlastitih baza podataka poznatih poliketidnih i peptidnih supstancija (CSDB) te potpuno novih poliketidnih i peptidnih supstancija produkata rekombinacije (r-CSDB). Ti će se produkti rekombinacije moći upotrijebiti za izbor supstancija s potencijalnom biološkom aktivnošću pomoću računalom vođenog dizajna lijekova u uvjetima in silico. Primjenjivost programskih paketa ClustScan i CompGen dokazana je u analizi genomskih sekvencija prokariotskih i eukariotskih mikroorganizama što žive u tlu, analizi metagenomske skupine podataka u uzorku iz morske vode, a i na nedavno opisanom primjeru \u27zajedničkog metaboličkoga puta\u27 u mikrobnog endosimbionta morske životinje

Untersuchungen zu Polyketidsynthasen aus Dictyostelium discoideum

Der in der vorliegenden Arbeit untersuchte Organismus Dictyostelium discoideum ist eine ubiquitär vorkommende Amöbe mit der Fähigkeit bei Nahrungsmangel in einen vielzelligen Fruchtkörper zu differenzieren. Bei der Sequenzierung des Genoms wurden 40 funktionale Gene für Polyketidsynthasen (PKS) beschrieben. Damit einhergehend wird ein großes Potential für die Biosynthese von Polyketiden vermutet, welches die bisher in D. discoideum beschriebenen Naturstoffe weit übersteigt. Der OSMAC-Ansatz (one strain-many compounds) wurde herangezogen um die Biosynthese von Sekundärmetaboliten zu aktivieren. Dabei wurden unter Verwendung verschiedener Extraktionsmittel mittels HPLC-DAD keine aktivierenden Bedingungen identifiziert. Unter Verwendung von RT-qPCR wurde pks26 als entwicklungs- und cAMP-abhängig exprimiertes Gen identifiziert. Um das PKS26-Biosyntheseprodukt zu identifizieren, wurden mittels homologer Rekombination Knock-Out- sowie Genaktivierungsstämme erzeugt. Ein Vergleich der Gesamtkultur-Extrakte von Wildtyp, Knock-Out- und Genaktivierungs-stamm mittels UHPLC-ESI-HRAM/MS und anschließender Hauptkomponentenanalyse des Gesamtmetaboloms führte zur Identifikation von 21 Kandidaten für das Biosyntheseprodukt von PKS26. Im Cheating-Assay wurde das Verhalten der pks26-Mutanten während der chimären multizellulären Entwicklung untersucht. Der publizierte Loser-Phänotyp der pks26-REMI-Mutante (restriction enzyme-mediated integration) konnte nicht bestätigt werden. Während der klonalen multizellulären Entwicklung verhielten sich die pks26-Mutanten wie ihr Elternstamm und zeigten in verschiedenen Fitnesstests keine Auffälligkeiten