Screening of a metagenomic library of Pseudoalteromonas tunicata for the identifications of genes involved in the production of bioactive compounds

The thesis is the result of one-year project carried out in the laboratory of University of New South Wales, Sydney, Australia. The project main aim was to develop an hight-throughput assay for the identification of bioactive compounds using the screening of a metagenomic library of E. coli clones. The bacterial genus Pseudoalteromonas contains numerous marine species, which synthesize biologically active molecules. Many Pseudoalteromaonas species have been demonstrated to produce an array of low and high molecular weight compounds with antimicrobial, algicidal, neurotoxic and other pharmaceutically relevant activities. P. tunicata is the best studied species within the genus. It lives in association with sessile eukaryotes such as algae and tunicates, and is a known producer of several bioactive compounds with activities towards other surface colonisers including bacteria, fungi, invertebrate larvae, diatoms, algal spores and protozoa. The aim of this study is to identify the gene(s) involved in the synthesis of bioactive compounds in the marine bacterium P. tunicata that act against eukaryotic organisms. The nematode Caenorhabditis elegans was used in this study as a model eukaryote for screening bioactive compounds produced by P. tunicata. The development of a screen of a genomic library of P. tunicata DNA allowed for the identification of genes encoding for compounds acting against C. elegans. Three positive clones with anti-nematode activity were found and there gene contents where analysed by genomic analysis and through random transposon mutagenesis. The genes involved in the anti-nematode activity encode for a novel fast-killling protein, moreover a gene cluster for the biosynthesis of a small molecule was found out to be involved in the slow-killing activity. Il batterio marino Pseudoalteromonas tunicata vive associato alla superficie di alghe e tunicati marini, possiede una pigmentazione verde-nera e produce una gamma di composti che inibiscono il fouling di larve di invertebrati, batteri, funghi e spore algali. Lo scopo di questo lavoro di tesi è stato lo sviluppo di uno specifico saggio anti-nematode ad alta risoluzione che permetta di individuare il gene responsabile della sintesi del composto anti-nematode e/o ricercare un suo possibile ortologo la cui attività sia nota nei genomi di altri organismi. Il saggio anti-nematode è stato realizzato attraverso lo screening di una libreria genomica, utilizzando come strumento di screening Caenorhabditis elegans. La libreria genomica utilizzata è stata creata inserendo un largo inserto di DNA di P. tunicata in Escherichia coli. Tre cloni positivi anti-nematode sono stati isolati attraverso lo screening della libreria, gli inserti di DNA sono stati estratti, sequenziati e trovata la loro posizione all’interno del genoma di P. tunicata. Gli inserti di DNA sono stati quindi reinseriti all’interno di E. coli creando una libreria di cloni knock-out, ognuno dei quali avesse una porzione dell’inserto di DNA silenziato, ed effettuando nuovamente lo screening di questa libreria contro C. elegans per identificare quale fosse il gene o i geni responsabili della sintesi del composto anti-nematode. I geni sono stati identificati e caratterizzati. Sono state, inoltre, saggiate le capacità anti algali in un saggio anti-diatomee utilizzando P.tunicata wild type e mutanti knock-out (nella produzione di pigmenti) allo scopo di comprendere la correlazione tra attività anti algali e la produzione di pigmenti in P. tunicata

Hydrogen peroxide-mediated killing of Caenorhabditis elegans by Enterococcus italicus and Lactococcus garvieae isolated from food

In this study, we used the nematode Caenorhabditis elegans as a model to assess the pathogenic potential of two species isolated from food, Enterococcus italicus and Lactococcus garvieae, for which few indications on pathogenicity are available. We identified the conditions under which E. italicus and L. garvieae are able to kill the nematode and suggest that the production of hydrogen peroxide (H2O2) by these two bacteria was involved in the death of C. elegans in our model system. The efficacy of E. italicus and L. garvieae to kill C. elegans differed, most likely related to each species' distinct ability to accumulate H2O2 (4.9 mM and 0.9–1.1 mM, respectively). Genome analysis of both species revealed that the genome of E. italicus contains a gene encoding a NADH oxidase which shows high amino acidic similarity with H2O2 -forming NOX-1 enzymes, while that of L. garvieae contains a gene codifying for a water-forming NADH-oxidase (NOX-2). Reverse transcriptase-PCR experiments carried out in presence of flavin adenine dinucleotide (50 mM) confirmed the presence of the two different genes and likely explains the different toxicity of E. italicus and L. garvieae against C. elegans in our study. The results obtained show for the first time the production of H2O2 in E. italicus and L. garvieae and indicate its toxic effect in the nematode C. elegans

Combining microbial functional metagenomics and Caenorhabditis elegans genetics to uncover and characterise novel bioactive compounds

Marine bacteria produce a wide array of biologically active metabolites involved in defence strategies and pathogenesis against a range of metazoans target organisms. In return bacteriovorous metazoans, including nematodes, have developed sophisticated strategies to neutralise or avoid such bioactives. The overall objective of this study was to investigate the antagonistic activities of bacteria-eukaryote interactions.The first aim of this thesis was to develop a novel functional (meta) genomic screen for the identification of inhibitors that target the nematode Caenorhabditis elegans. Environmental DNA from marine habitats was expressed in Escherichia coli and the resulting functional (meta) genomic libraries were screened by selective grazing of the nematode C. elegans, in a simple, rapid, high-throughput manner.Next, this project aimed at characterizing antagonistic activities that target C. elegans and study their toxic effect in the nematode. Genetic and microscopic analysis uncovered known and novel bioactive compounds including the small molecules tambjamine and violacein, which appear to facilitate bacterial colonisation and induce apoptosis in the nematode. An array of genes and gene products involved in antinematode activities was also identified including a gene encoding for a novel protein involved in a fast killing activity.Finally, this study investigated the sophisticated strategies that the nematode mounts to neutralize or avoid bacterial toxic compounds. The role of both C. elegans behavioural and immune system strategies in mediating the nematode defence in response to toxic bacterial compounds were investigated. In the nematode, a complex behavioural strategy known as aversive learning is employed to avoid violacein toxicity. When noxious violacein cannot be avoided, the toxic compound appears to activate the insulin-like signaling cascade, an evolutionary conserved innate immunity pathway.In summary, this work combines functional (meta) genomics and C. elegans genetics to identify bioactive compounds from marine bacteria and uncover animal defence strategies in response to bacterial secondary metabolites

Assessing the Effectiveness of Functional Genetic Screens for the Identification of Bioactive Metabolites

A common limitation for the identification of novel activities from functional (meta) genomic screens is the low number of active clones detected relative to the number of clones screened. Here we demonstrate that constructing libraries with strains known to produce bioactives can greatly enhance the screening efficiency, by increasing the “hit-rate” and unmasking multiple activities from the same bacterial source

Wax hearts: seeking the antiquity of cardiac pathology

Wax models of normal and diseased organs were formerly essential medical teaching tools. The ceroplastic heart models from two 19th century pathology museums at the Universities of Florence (n = 8) and Coimbra (n = 10) were analysed. The Florentine collection comprised congenital malformations as well as infectious and inflammatory disorders. The Coimbra waxworks included congenital defects, cardiac hypertrophy and dilation, valvular pathology and cardiac adiposity. This study focuses on heart diseases and teaching resources in European university hospitals during the 19th century. It also highlights the importance of wax models in medical education both then and today, in an era of informatics and digital photography

Correction: Antinematode Activity of Violacein and the Role of the Insulin/IGF-1 Pathway in Controlling Violacein Sensitivity in Caenorhabditis elegans.

[This corrects the article DOI: 10.1371/journal.pone.0109201.]

Antinematode activity of Violacein and the role of the insulin/IGF-1 pathway in controlling violacein sensitivity in Caenorhabditis elegans.

The purple pigment violacein is well known for its numerous biological activities including antibacterial, antiviral, antiprotozoan, and antitumor effects. In the current study we identify violacein as the antinematode agent produced by the marine bacterium Microbulbifer sp. D250, thereby extending the target range of this small molecule. Heterologous expression of the violacein biosynthetic pathway in E. coli and experiments using pure violacein demonstrated that this secondary metabolite facilitates bacterial accumulation in the nematode intestine, which is accompanied by tissue damage and apoptosis. Nematodes such as Caenorhabditis elegans utilise a well-defined innate immune system to defend against pathogens. Using C. elegans as a model we demonstrate the DAF-2/DAF-16 insulin/IGF-1 signalling (IIS) component of the innate immune pathway modulates sensitivity to violacein-mediated killing. Further analysis shows that resistance to violacein can occur due to a loss of DAF-2 function and/or an increased function of DAF-16 controlled genes involved in antimicrobial production (spp-1) and detoxification (sod-3). These data suggest that violacein is a novel candidate antinematode agent and that the IIS pathway is also involved in the defence against metabolites from non-pathogenic bacteria

Nematode killing assay and dose response assay (N2 animal vs the alive and heat killed 20G8 clone and D250 strains).

<p>(A) Killing kinetics of <i>Microbulbifer</i> sp. D250 and dV2 mutant deficient in violacein production. Negative control OP50 is a non-pathogenic strain of <i>E. coli</i>. (B) Dose response of <i>C. elegans</i> to pure violacein added to 20G8<i>vioA⁻</i> mutant bacteria alive and heat killed. Each point on the graph represents the average survival of worms after seven days exposure to violacein (C) Kinetics of nematode killing when nematodes are fed either the live or heat killed 20G8 clone or 20G8<i>vioA⁻</i> mutant. Each data point represents means ± the standard error of three replicate plates. <i>p</i> values were calculated on the pooled data of all of the plates in each experiment by using the log-rank (Mantel–Cox) method.</p