Exploring the undiscovered microbial diversity on marine surfaces
A project undertaken at the University of New South Wales under the supervision of Dr Suhelen Egan, Dr Carola Holmström and Dr. Torsten Thomas
Prokaryotic micoorganisms compromise a large portion of the organic biomass of the world’s ocean and play an important role in essential biogeochemical cycles and food webs in this ecosystem (Cho & Azam 1990; Fuhrmann & Sleeter, 1980). In particular surface colonisation by microorganisms is ubiquitous in marine systems with a large proportion of microbes occurring as complex communities. Often the negative impacts of microorganisms on humanity occur at surfaces- be it the surfaces of seafood (fish, oysters etc.) or fouling of ships’ hulls (Holmström & Kjelleberg, 2000; Lappin-Scott & Costeron, 1989; Lewis, 1994; Marshall, 1994). However despite their importance comparatively little is known about the phylogenetic composition of these complex, microbial population and the functional roles of their members.
Living surfaces are ideal system in which to explore colonisation by microorganisms because eukaryotes are subject to a constant bombardement from the millions of microbial cells typically found in a millilitre of seawater. Unlike inanimate surfaces the focus of most research on living surfaces provide an interactive system in which to study colonisation and communication between eukaryotes and prokaryotes (Kjelleberg & Steinberg, 2002).
Our research on microbial colonisation of living marine surfaces has focused on two model systems:
In general the methods used to date for assessing diversity in any environments have provided conflicting results (i.e. little overlap between cultured microbes and those found using non-culture dependent studies) and indicate that a) we do not have a complete picture of the microbial diversity in the marine environment and that b) we have little information about the functional properties of the large portion of uncultured organisms. This is consistent with estimates for other microbial system, which indicate that less than 1% of microbes have been successfully cultured and studied in the laboratory (Rappe & Giovannoni, 2003). Two factors have been widely recognised as contributing to the limited success in studying microbial diversity. Firstly, traditional culturing techniques are imperfect in mimicking the biological, physical and chemical conditions encountered by the microbe in the environment (Rappe & Giovannoni, 2003). As such culturing will favour and isolate those organisms that are best suited or adapted to the specific laboratory conditions applied. Examples of these include fast-growing organisms that rapidly form colonies on agar plates and hence overgrow or suppress other microbes. Secondly, culture-independent phylogenetic approaches rely extensively on the “universal” character of the 16S rDNA specific primer i.e. the “universal” primers are assumed to capture everything including the unknown diversity. It has now been realised that these primers have a preference to amplify certain sequences and as such provide a skewed picture of the actual microbial diversity (Suzuki & Giovannoni, 1996). In addition, recent random DNA sequencing of environmental DNA from the Saragasso Sea has identified 16S rDNA sequences that would not be (or very inefficiently) detected with the current 16S rDNA primers (Venter et al., 2004).
Recently an alternative approach to study microbial diversity has been successfully established (Liles et al., 2003; Suzuki et al., 2004). This involves the isolation of total microbial DNA from the environment, which is then cloned in large fragment into E coli. Technological advances in environmental DNA isolation and purification in addition to the development of efficient cloning systems (e.g. bacterial artificial chromosomes (BACs) that can harbour up to 350 000 basepairs) have started a field that is now termed environmental genomics. In principle, this approach allows us to study microbial and genetic diversity of environmental communities without culturing or PCR-based amplification. The power of this technology is illustrated by the fact that just 10 BACs clones in E. coli can cover the genetic information of a specific uncultured organism. As such BAC libraries with 10 000 or more clones can harbour and characterise the genomes of an entire microbial community.
The aim of this project is to explore the communities of microorganisms on living surfaces and to understand the formation of these communities on seaweeds. Specifically we are using novel genomic approaches to determine the composition of microbial communities and their function on the surfaces of the model seaweeds D. pulchra and Ulva sp.
To assess the bacterial community we will construct BAC libraries and analyse these for gene and species diversity (through analysis of marker genes such as the16S rDNA and associated functional genes). This will be complemented with direct analysis of abundance and distribution of selected groups on the algal surface. Specifically we will:
It is expected that this research will result in a comprehensive understanding of the bacterial community associated with the surface of two model marine algae. Not only will this approach identify new members of this community but by analysis of specific clones we will gain knowledge of the functional properties of the members of this surface-community. In addition we plan to use the BAC libraries in future studies to screen for novel functional properties such as new antibiotics and bioactives. The discovery of novel bioactive compounds has clear benefits to human health and the environment.
Burke, C, Thomas, T, Egan, S and Kjelleberg, S (2007). The use of functional genomics for the identification of a gene cluster encoding for the biosynthesis of an antifungal tambjamine in the marine bacterium Pseudoalteromonas tunicata. Environmental Microbiology 9 (3), 814-818. (View PDF doc)