s.kjelleberg@unsw.edu.au

The microbiology based research programs at the Centre for Marine Bio-Innovation (CMB) focus on 1) Bacterial Biofilm and Cell-Cell Signalling, 2) Diversity and Bioactivities of Marine Microbial Communities, and 3) Bioremediation. Honours projects are offered in each of these research clusters. The projects include strong links with international research and industrial partners. For further information on the CMB go the www.cmb.unsw.edu.au.

Program 1:   Bacterial Biofilms and Cell-Cell Signalling

Bacteria form biofilms on all surfaces. Biofilms are essential in most ecosystems processes and they are of great benefit in many applications. However, they also lead to many problems, with poor process performance and corrosion of material in industrial settings, and in the medical context where biofilms harbour pathogens. In fact, more than 60% of all bacterial infections are estimated to be a result of biofilm formation. It is now understood that biofilm formation takes place in distinct stages, involves highly specific differentiation events, involves a central role for cell-cell quorum sensing signaling, and features a unique gene expression and the formation of specialised cells. A strong feature of the CMB biofilm program is the development of novel, environmentally friendly, biofilm control technologies. In particular, such projects focus on the use of quorum sensing blockers and small signaling molecules for inducing differentiation and hence dispersal of biofilms

Projects include, but are not limited to

Increased virulence, survival and fitness of mixed bacterial communities

Genetic change underpins the ability of organisms to adapt to changing environments.  Recent data from our research has demonstrated that bacterial biofilms are active sites of genetic change in bacteria, leading to the formation of genetic variants with altered traits, eg. changes in virulence factor production, altered biofilm formation and stress survival.  In experiments where we have compared the virulence and survival of combinations of variants with non-variant populations, we have shown that the mixed community is superior.  This project will compare the virulence and fitness of Pseudomonas aeruginosa variants derived from clinical and environmental specimens and test the Hypothesis that the activity of the mixed community is greater than the sum of its individual parts.  This is a leading edge concept in evolution and infection, with significant implications for Treatment of Infectious Diseases, Environmental Survival of bacteria, and Community based Evolution.  The project will be approached using a combination of different techniques including:  reporter gene expression, DNA arrays, proteomics, PCR, Confocal Microscopy, Tissue culture, and the Caenorhabditis elegans virulence model. 

Regulation of biofilm formation and anti-protozoal activity of Vibrio cholerae

Vibrio cholerae is the causative agent of cholera and infects hundreds of thousands of people every year.  In between outbreaks, it is thought to exist in environmental reservoirs within biofilms, where V. cholerae must protect itself from the major stress on bacteria which is predation by protozoa.  Virulence factors expressed by bacterial pathogens may have evolved in the environment where bacteria must defend themselves against predation by protozoans.  We have demonstrated that bacteria attach to surfaces and form biofilms in the presence of protozoans, where bacterial cells in the biofilms are protected from grazing. V. cholerae appears to actively respond to the presence of protozoans by increasing biofilm formation and the expression of virulence factors.  This project uses the recently described recombination-based in vivo expression technology (RIVET) in order to identify genes that are expressed in the presence of grazing pressure.  Identification of genes expressed in response to grazing pressure will allow us to understand how virulence evolves in nature.

Natural selection operates at different levels of biological complexity, including genes, single cells and multicellular organisms. Whilst it is generally accepted that natural selection also acts on populations of organisms as an evolutionary unit, most researchers deny the possibility that communities or multi-species mixtures of populations have enough identity to be selected for or against. We have developed two experimental systems using different bacteria and protozoa to probe the boundary between group and community level selection. This project asks if individuals make sacrifices for the good of a community.

The specific aims of this project are to

1. Learn how to run chemostats and handle and quantify a selection of bacterial strains and protozoa. Develop protocols for transferring cultures from one chemostat to another.  2. Establish and maintain mixed species communities of bacteria and protozoa and to monitor the evolution of bacterial growth rates in real time. 3. Explore the benefits of reduced growth rates in competition experiments between different bacterial strains.

Signaling pathways involved in biofilm development and dispersal

Biofilm formation and dispersal is a succession of diverse and complex regulatory events. In addition, biofilms are considered to be the predominant form of microbial life in nature. This has dramatic consequences especially for clinical environments, because residing in biofilms has been shown to confer increased resistance to bacterial cells to biocides. Thus, the understanding of environmental signals, regulatory mechanisms, and the corresponding expression of target genes involved in this developmental process is crucial for biofilm control strategies. Only recently, the novel, intracellular signaling molecule cyclic di-guanosinmonophosphate (c-di-GMP) has been found to play a key role in regulating biofilm development in response to environmental conditions. This project will investigate the impact of c-di-GMP signaling involved in biofilm formation and dispersal in the opportunistic pathogen Pseudomonas aeruginosa. The goal of this project is to gain further insights in specific c-di-GMP dependent processes, using model systems which have been identified and established in our laboratory. The spectra of methods will cover a wide range of techniques such as reporter gene expression, random and site-directed mutagenesis, PCR, cloning, bacterial two-hybrid systems, enzymatic tests, virulence assays, and competition experiments.

Research fellows involved in the biofilm and signaling program are Nicolas Barraud, Janosch Klebensberger, Diane McDougald, Scott Rice, and Mike Manefield.

Program 2:  Genomics and Bioactivities of Marine Microbial Communities

Micoorganisms make up a large portion of the organic biomass in the world’s ocean and are essential biogeochemical cycles and food webs. In particular, surface colonisation by microorganisms is ubiquitous in marine systems with a large proportion of microbes occurring as complex communities. However despite their importance comparatively little is known about the phylogenetic diversity of these complex, microbial populations and the functional roles of their members.

Surfaces of marine eukaryotes (“living surfaces”) are ideal systems to explore colonization by microorganisms as they are subject to a constant bombardment from the millions of microbial cells typically found in a millilitre of seawater. Our research on colonization of living surfaces of algae and sponges is an interactive system in which to study communication between eukaryotes and microoganisms and provide a rich source of novel bioactives with medical and industrial applications.

Projects include, but are not limited to

Marine sponges harbour a rich diversity of microbial cells, that can make up to 50% of their body mass. These prokaryotic symbionts are thought to perform essential functions for the sponge including waste removal and nutrient acquisition, yet are also able to evade being consumed through phagocytosis by the sponge cells. However due to the difficulty in culturing many of these prokaryotic organisms, the nature of these symbionts and their interaction with the sponge host is poorly understood.

We have recently started a metagenomic project to define the functional diversity of symbionts in the sponge Cymbastela concentrica. While this will provide a unique insight into the genetic potential of the symbionts, we now need to understand if the functional properties are being utilised or expressed. For this we will use whole-community proteomics (metaproteomics) to measure the in situ expression of hundreds and thousand of protein in the sponge symbionts. The resulting expression profile and the identity of the proteins will give us a comprehensive picture of the functional activities and roles of microbes in sponges.

1. To establish efficient protein extraction procedures for whole microbial communities associated with the sponge Cymbastela concentrica. 2. To determine the protein profile of the sponge symbionts with modern peptide mass-spectrometry. 3. To link the protein profile with metagenomic sequence information to assign protein expression to specific organisms. 4. To build a comprehensive picture of microbial activities and roles in the sponge Cymbastela concentrica.

Genomic studies of a model marine surface associated bacterium

The marine surface associated bacterium Pseudoalteromonas tunicata is a model organism on which to study the production and regulation of bioactive metabolites. This organism is known to produce a number of compounds with both specific and general affects against a variety of bacteria and eukaryotes. However to date the exact nature of several of these compounds remains unclear as does the environmental parameters that lead to their production.  The recently sequenced genome provides many insights into the potential mechanisms of interaction between P. tunicata and it’s marine eukaryotic hosts (algae and invertebrates).

The overall aim of this project is to determine mechanisms by which P. tunicata mediates interactions with marine eukaryotic host and establishes itself as a successful member of the marine microbial surface community.

The specific aims of this project include

Members of the alpha-proteobacterial Roseobacter/ Ruegeria clade (RR) are widespread in the marine ecosystem and play unique roles ranging from primary energy production to sulfur compound cycling to pathogens of higher organisms. RR species have been described as important members of planktonic communities as well as powerful colonisers and biofilm formers of living surfaces.

Bacteria can initiate a dispersal event from within biofilms with cells swimming towards new, uncolonised surfaces. For some gamma-proteobacteria it has been shown that dispersal cells have stable phenotypic properties caused by genetic mutations which give the dispersal population an improved fitness to colonise new habitats. However it is unclear if this phenomenon is common in the bacterial domain or what mechanisms are responsible for the genetic variation.

The specific aims of this project are

1. To investigate the variation of dispersal cells for phenotypic properties such as pigmentation, quorum sensing, motility and substrate utilisation. 2. To use the genome information of R. gallaeciensis 2.10 to identify candidate genes involved in the phenotypic variation. 3.  Use PCR and DNA sequencing to investigate mutation in the candidate genes for the observed variants. 4.  Based on the mutational events observed build a model for the generation of genomic variation in the R. gallaeciensis 2.10 dispersal population.

Research fellows:  Suhelen Egan, Torsten Thomas, Carola Holmstrom

Program 3:   Bioremediation

Electron shuttles for degradation of chlorinated pollutants

The global pandemic of cancers and degenerative diseases are increasingly being linked to soil, water and air pollution. Sydney, like all major cities around the world, is home to sites polluted with chlorinated compounds (Botany Bay, Sydney Harbour, Homebush Bay). Whilst many bacteria have been identified to break down several chlorinated compounds, there remain many compounds for which there are no known bacteria that can degrade them. We have identified molecules, known as electron shuttles that enable bacteria to degrade previously recalcitrant chlorinated compounds. This project involves testing electron shuttles and bacteria for their ability to degrade key pollutants in the Sydney area.

The specific aims of the project are to

1.  Use simple abiotic dechlorination assays, to test the ability of electron shuttles to degrade pollutants commonly found in the Sydney region. 2. Screen bacterial isolates from a range of environments for their ability to reduce electron shuttles identified in the first aim. 3. Assess the impact of electron shuttles on the growth of active isolates identified in the second aim. 4. Test active electron shuttles and bacterial isolates on the degradation of key pollutants.

Characterisation of solvent tolerant microbes from bioremediation

1. Set up and monitor aerobic and anaerobic enrichment cultures to select for solvent tolerant organisms 2.  Identify species in the solvent tolerant microbial communities through clone library construction and DNA sequencing and attempt to isolate solvent tolerant species using traditional microbiological techniques. 3.  Characterise the response of solvent tolerant enrichment cultures and pure cultures to different solvents at different concentrations and assess their utility in the degradation of carbon tetrachloride and perchloroethene.