Screening of a genomic library of Pseudoalteromonas tunicata for the identification of genes involved in the production of bioactive compounds.

 Abstract 
 
 
The bacterial genus Pseudoalteromonas contains numerous marine species, which 
synthesize biologically active molecules. Many Pseudoalteromaonas species have 
been demostrated 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. 
 
 
 1 Introduction 
 
 
1.1 Novel bioactive compounds 
 
The intense competition among environmental microbes for limited resources and 
nutrients is providing a strong ecological reason for why some organisms produce 
antimicrobial "secondary metabolites". These molecules, although not directly 
required for growth in the laboratory, maybe essential for the microbes in the 
environment as they confer an advantage in the competition for nutrients and living 
space. Many microbes even compete with organisms outside their domain and hence 
produce antifungal drugs, herbicidal agents, 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 
development of drugs, new biocatalysts and enzymes, which work under varied 
conditions of temperature and pH. There is  also a substantial 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). Pharmaceutical industries are 
interested in high-throughput assays to screen for inhibitory compounds against 
bacterial pathogens of human and/or breeding animals. 
The search for novel bioactive compounds has traditionally focused on terrestrial 
organisms; however, during the last thirty years interest has shifted towards the 
marine environment (Faulkner, 2000; Zhang, 2005).  Marine organisms are rich in 
bioactive compounds, which are believed to have evolved as chemical defence 
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 mechanisms in highly competitive environments (Engel, 2002; Zhang, 2005).  
Hundreds of new compounds from marine organisms are isolated every year and 
have been reported to have for example antibacterial, antifungal, cytotoxic, 
neurotoxic, immunosuppressive, antiviral, and antiinflammatory activities (Blunt 
2005; Faulkner, 2002). 
Microbial diversity in the environment is now recognised to be much greater than 
previously estimated (Pace, 1997) and many novel marine microorganisms have now 
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, microbial 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 such as algae and marine invertebrates 
(Holmstr m and Kjelleberg, 1999).  The genus Pseudoalteromonas has received 
significant attention in field of  natural product chemistry and microbial ecology 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 other pharmaceutically relevant activities (Holmstr m et al., 
2002; Holmstr m et al., 1999; Kalinovskaya et al., 2004; Longeon et al., 2004; 
Lovejoy et al., 1998; Hansen et al., 1965; Bowman, 2007). Compounds produced 
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 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). 
Pseudoalteromonas species appear to be ecologically significant and to date have 
been shown to influence biofilm formation in various marine econiches, to have 
predator-like interactions within the microbial loop, influence the settlement, 
germination and metamorphosis of various invertebrate and algal species and also to 
potentially 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 their novel natural 
products as well as ecological connections in the marine ecosystem remain to be 
made (Bowman, 2007).  
Studies by Holmstr m (Holmström et al., 1996) found pigmented strains from a 
collection of marine bacterial isolates were effective in the inhibition of the 
settlement of various fouling invertebrates and algae. A subsequent analyses of 
some of these isolates lead to the general conclusion that pigmented 
Pseudoalteromonas species possess a broad range of bioactivities associated with 
the secretion of extracellular compounds, several of which include pigment 
compounds (Holmstr m, 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 (Holmstr m 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 
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 bacterium a dark green appearance (Franks et al., 2005). P. tunicata produces 
several bioactive compounds with directed activities towards specific organisms 
including bacteria, fungi, invertebrate larvae, diatoms, algal spores and protozoa 
(Holmstr m et al., 2002; Holmstr m 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, the time at 
which 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 (Holmstr m, et al., 1992).. 
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-Fr hner et al., 2003; Dur n et al., 
1983; Dur n, 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 
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 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 and functional screening have identified a gene cluster involved in 
tambjamine biosynthesis and antifungal activity (Egan et al., 2002, Burke et al. 
2007). 
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, and the antifungal 
tambjamine (Burke et al. 2007) however the genes and biosynthetic pathways of 
the 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. Furthermore the P. tunicata genome was recently sequenced, which 
allows 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 and widely used 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 nematode is sensitive to a number of charged and 
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