1 Introduction
1.1 Novel bioactive compounds
The intense competition among soil microbes for limited nutrients is thought to be a major reason why some of these organisms produce antimicrobial "secondary metabolites"— these molecules, although not directly required for growth, maybe useful for environmental microbes, since confer an advantage in the competition for nutrients and living space. However, soil microbes make several natural products other then antibacterials, which include anticoccidial agents, antifungal drugs, herbicidal agents, anticancer drugs, insecticidal, nematocidal agents, and enzyme inhibitors (Osburne et al., 2000).
Microbial bioactive compounds and their encoding genes have applications in many different fields. Natural compounds and their derivatives have an important role in discovery of drug, new biocatalysts and enzymes which work under varied conditions of temperature and pH are also of interest for food production, synthetic chemistry and industrial biotechnology (Lorenz and Eck, 2005). Compounds which prevent or inhibit the fouling of marine surfaces could result in significant financial savings to the shipping and aquaculture industries (Braithwaite and McEvoy, 2005; Yebra et al., 2004). Alimentary industries are interested in high-throughput assay useful to screen for harmful compounds from bacterial strain pathogenic for human and/or for breeding animals.
focused on terrestrial organisms; however, during the last thirty years it has seen an explosion of interest in the marine environment (Faulkner, 2000; Zhang, 2005). Marine organisms are rich in bioactive compounds which are believed to have evolved as chemical defence mechanisms in highly competitive environments (Engel, 2002; Zhang, 2005). Hundreds of new compounds from marine organisms are isolated every year, which are reported to have activities including antibacterial, antifungal, cytotoxic, neurotoxic, immunosuppressive, antiviral, and antiinflammatory (Blunt 2005; Faulkner, 2002).
Microbial diversity is now thought to be much greater than previously estimated (Pace, 1997) and many novel marine microorganisms have recently been identified which belong to both previously described as well as completely new phyla (Schleper et al., 2005; Stach et al., 2005; Venter et al., 2004). The marine environment contains a vast array of virtually unexplored microbial biodiversity which could potentially yield many novel bioactive compounds.
1.2 The species Pseudoalteromonas tunicata as a source of bioactive compounds.
Pseudoalteromonas genus belongs to the class Gammaproteobacteria. Bacteria from this genus are found exclusively in marine environments and were found in association with higher organisms as algae and marine invertebrates (Holmström and Kjelleberg, 1999). The genus Pseudoalteromonas has come to attention in the natural product and microbial ecology science fields in the last decade. Pigmented species of the genus have been shown to produce an array of low and
high molecular weight compounds with antimicrobial, anti-fouling, algicidal, neurotoxin and various pharmaceutically relevant activities (Holmström et al., 2002; Holmström et al., 1999; Kalinovskaya et al., 2004; Longeon et al., 2004; Lovejoy et al., 1998; Hansen et al., 1965; Bowman, 2007). Compounds formed include toxic proteins, polyanionic exopolymers, substituted phenolic and pyrolle-containing alkaloids, blockers of voltage-gated, Na+ ion membrane channels as tetrodotoxin, cyclic peptides and a range of bromine-substituted compounds (Gallacher and Birkbeck, 1993; Simidu et al., 1990; Bowman, 2007). Ecologically, Pseudoalteromonas appears significant and to date has been shown to influence biofilm formation in various marine econiches; involved in predator-like interactions within the microbial loop; influence settlement, germination and metamorphosis of various invertebrate and algal species; and may also be adopted by marine flora and fauna as defensive agents. Studies have been so far limited to a relatively small subset of strains compared to the known diversity of the genus suggesting that many more discoveries of novel natural products as well as ecological connections these may have in the marine ecosystem remain to be made (Bowman, 2007).
Studies by Holmström (Holmström et al., 1996) found deeply pigmented strains within a marine bacterial isolate collection were effective in the inhibition of the settlement of various fouling invertebrates and algae. Based on subsequent analyses some of these isolates lead to the general conclusion that pigmented
Pseudoalteromonas species possess a broad range of bioactivity associated with the secretion of extracellular compounds, several of which include pigment compounds (Holmström, and Kjelleberg 1999).
One of the most extensively studied species within this genus is P. tunicata. This species possesses the highest and broadest range of anti-fouling activities observed to date and was originally isolated from the surface of an adult tunicate off the coast of Sweden (Holmström et al., 1998). This bacterium has also been isolated from the surface of the marine algae Ulva lactuca off the east coast of Australia (Egan et al., 2000). P. tunicata produces at least two pigments, these include a yellow (tambjamine) and a purple (violacein) pigment which, when combined, give the bacterium a dark green appearance (Franks et al., 2005), and produces several bioactive compounds with directed activity towards specific organisms including bacteria, fungi, invertebrate larvae, diatoms, algal spores and protozoa (Holmström et al., 2002; Holmström and Kjelleberg 1999). Production of bioactive compounds by P. tunicata is believed to aid in surface colonisation and contribute to the prevention of biofouling of the living marine surfaces with which it is associated (Egan et al., 2000)
Antibacterial activity in P. tunicata has been linked to the production of a 190 kDa antibacterial protein designated AlpP. This protein is active against a range of Gram negative and Gram positive bacteria. P. tunicata cells in stationary phase, which is when the protein is produced, appear resistant to the effects of AlpP (James et al., 1996)
The compound responsible for the inhibition of algal spore germination has been identified as a polar, heat sensitive molecule between three and 10 kDa in size (Egan et al., 2001).
Inhibition of larval settlement has been shown to be due to a small (less than 500 Da), heat stable polar compound (Holmström, et al., 1992). Due to the difficulty
of purifying water soluble compounds no further characterisation has been carried out and the genes responsible for their production are as yet unknown.
The deep purple pigment produced by P. tunicata has been identified as violacein (Franks, 2005) a pigment originally isolated from Chromobacterium violaceum. Violacein from C. violaceum has been reported to have activities including antibacterial, cytotoxic, antiviral and is also believed to relieve grazing pressure due to its toxicity to nanoflagellates (Andrighetti-Fröhner et al., 2003; Durán et
al., 1983; Durán, and Menck 2001; Matz et al., 2004). While the violacein produced by P. tunicata displays anti-flagellate activity, other activities due to the production of violacein have not been observed in this bacterium.
Antifungal activity in P. tunicata has been linked to the expression of a yellow pigment (Egan et al., 2002), which has been recently identified as a tambjamine and designated YP1 (Franks et al., 2005).
Tambjamines have previously been isolated from marine invertebrates such as nudibranchs, bryozoans and ascidians (Blackman and Li, 1994; Lindquist and Fenical 1991). Only one other tambjamine compound has been isolated from a bacterial source, BE-18591 from Streptomyces sp. BE18591.
This compound is reported to have cytotoxic activity (Kojiri et al., 1993) while YP1 from P. tunicata is the first tambjamine reported to have antifungal activity (Franks, 2005). Mutagenesis studies have identified a gene cluster involved in tambjamine biosynthesis and antifungal activity (Egan et al., 2002).
While previous studies have provided valuable insight into the nature of bioactive compounds produced by P. tunicata, much is still unknown about the genetics involved. The gene encoding for the antibacterial protein AlpP has been
identified, as have the genes responsible for violacein biosynthesis, however the genes and biosynthetic pathways of the antifungal, antialgal, antilarval and antidiatom compounds are yet to be described.
The range of bioactive compounds produced by P. tunicata makes it a good model bacterium for the study of the detection and characterisation of bioactive compounds.
The P. tunicata genome was recently sequenced, this tool allow for more in-depth analysis of the genes responsible for the production of bioactive compounds in P.
tunicata.
1.3 C. elegans as a model organism to screen for bioactive compounds
.
Caenorhabditis elegans is a powerful genetic model to explore toxicology of bioactive compounds and/or to investigate bacterial pathogenicity. Its genome, and biosynthetic and metabolic pathways are highly studied, including most of the known components involved in cellular development, the nervous system, and cell death genes (Riddle et al., 1997; The C. elegans Sequencing Consortium, 1998; Nass and Blakely, 2003). The worm is sensitive to a number of charged and uncharged molecules that can easily penetrate its brain, including heavy metals, organic toxins, and a wide range of human neuroactive drugs (Rand and Johnson, 1995; Link et al., 2000; Nass and Blakely, 2003). C. elegans has a highly developed chemosensory system that enables it to detect a wide variety of volatile (olfactory) and water-soluble (gustatory) cues associated with food, danger, or other animals. Much of its nervous system and more than 5% of its genes are
devoted to the recognition of environmental chemicals. Chemosensory cues can elicit chemotaxis, rapid avoidance, changes in overall motility, and entry into and exit from the alternative dauer developmental stage, the stress resistant life stage of C. elegans (Bargmann, 2006). The nematode has also been utilized to screen for mutant proteins that have altered function in the presence of the drugs (Riddle
et al., 1997; Link et al., 2000).
C. elegans is also very useful as model hosts for bacterial infections, it is being used for approaches as diverse as testing the virulence of chosen pathogen mutants (Vaitkevicius et al., 2006; Blow et al., 2005), screening large banks of pathogen mutants for those with attenuated virulence (Miller and Neely, 2005; Styer et al., 2005) or dissecting the host mechanisms involved in pathogen invasion and intracellular replication (Cheng et al., 2005; Agaisse et al., 2005; Philips et al., 2005; Kurz and Ewbank, 2007).
Its small size (~1 mm), large number of progeny (300 to 1000 animals from a single hermaphrodite), quick generation time ( 3.5 days), and ease of growth in 96- or 384-well tissue culture plates, allow for rapid growth and ease of cultivation in a laboratory (Riddle et al., 1997; Nass and Blakely, 2003). C.
elegans also provides facile approaches to determining endpoints of toxicity or pathogenicity. If a specific behaviour is dependent on a particular cell type or molecular pathway, abnormal movement of the animals as observed under a dissecting microscope would be sufficient to determine toxicity (Riddle et al., 1997). Conversely if animal death is the endpoint, simple lack of movement of the animals following touching with a hair or a metal pick under the microscope could be sufficient.
C. elegans is maintained in the laboratory either on solid or in liquid growth medium.
They can be grown on agar plates using E. coli as a food source, or in liquid cultures with or without bacteria (Hope, 1999; Link et al., 2000). Worms grown on agar plates generally utilize one of two types of bacteria and media, depending on the experimental goal. Animals grown on bacteria OP50, a uracil auxotroph that forms a thin bacterial lawn when maintained on NGM minimal medium, are often used when examining particular phenotypes such as movement, lethal toxicity or cellular fluorescence. (Nass and Hamza, 2007)
C. elegans is not a target organism of the marine fouling, in this study the nematode was used as a powerful model organism for screening bioactive compounds produced by P. tunicata which is known to produce a wide range of well characterized bioactive compounds acting against fouling target and non-target organisms (Burke et al., 2007; Egan, 2001).
1.4 Approach with metagenomic analysis for the identification of genes encoding for bioacitve compounds.
Many natural products are bacterial and fungal secondary metabolites, but as most microorganisms cannot easily be cultivated, it is probable that many potentially active compounds have never been characterized. Metagenomics might have an invaluable role in the discovery process of new bioactive molecules (Daniel, 2004; Osburne et al., 2000).
characterised through pure culture methods. Studies typically involve growing up pure cultures, screening both supernatant and cell extracts for activity and random mutagenesis of the bacterium to elucidate the genes and biosynthetic pathways responsible for production of the compounds. While these methods provide valuable information they can be very slow and are limited to culturable bacteria. As such microbiologists are increasingly turning to molecular and culture independent methods of exploration and discovery (Courtois et al., 2003; Donadio
et al., 2002; Li and Qin, 2005, Zhang et al., 2005)
Metagenomics employs the use of DNA libraries for the study of collective microbial genomes from an environmental sample, this field has gained much popularity in the last five years as it allows access to the vast diversity of presently unculturable microorganisms (Handelsman, 2004; Riesenfeld et al., 2004). Metagenomic analysis involves isolating DNA from an environmental sample, cloning the DNA into a suitable vector, transforming the clones into a host bacterium, and screening the resulting transformants. Libraries are constructed from DNA extracted from an environment or from a prokaryotic or eukaryotic donor of interest and maintained in a heterologous host such as E. coli. The DNA contained in the library is then analysed either by sequence based or functional analysis. Libraries such as these have been used to study microbial diversity (Béjà et al., 200, Suzuki et al., 2004, Venter et al., 2004) and as a bioprospecting tool in the search genes encoding for novel bioactive compounds. Studies such as these have resulted in the identification of novel antibiotic compounds and novel biocatalysts and enzymes (Rondone et al., 2000; Song et
has also been used to identify the genes encoding the biosynthesis of known bioactive compounds isolated from higher organisms and to demonstrate that in fact these compounds are microbial in origin (Piel et al., 2004; Schmidt et al., 2000).
For both culturable and unculturable bacteria, large insert DNA libraries enable the rapid detection and characterisation of novel bioactive compounds and the genes from which they are produced. Large insert vectors such as fosmids, cosmids and BACs (bacterial artificial chromosomes) with the capacity to hold from 40 (fosmids and cosmids) up to 350 kb (BACs) of insert DNA allows for proteins and complexes encoded in large operons to be captured and expressed in a single heterologous host. Library clones can be rapidly screened for activity and active clones subjected to further studies such as DNA sequencing and transposon mutagenesis. This method effectively reduces the amount of DNA to work with from an entire genome to a relatively small fragment.
In this study we screened a genomic library with a large DNA insert of P. tunicata genome for anti-nematode activity.
1.5 Aim of this study
