Screening and Culturing


Jonathan Kennedy

Research Programmes for Senior Post Doctoral Fellows:

 

Jonathan Kennedy [email- j.kennedy@ucc.ie]

Metagenomic and Culture-based Approaches to Biodiscovery from Sponges

The main objectives of this research project are the utilisation of the sponge microbiota for the biodiscovery of novel bioactive agents and industrially useful enzymes.  Two main approaches are being adopted - a culture-based approach and, as the vast majority of the sponge microbiota are uncultured, a culture-independent or metagenomic approach. 

For the culture-based approach a collection of marine-sponge derived microorganisms are being cultured, catalogued, extracted and assayed for anti-microbial activities.  Following initial assays selected microbes are being studied in more detail to determine the antimicrobial spectrum of activity, the chemical structure of the antibiotic and the genetics of their biosynthesis.

The functional metagenomics approach will enable access to the biochemical and genetic diversity present in the uncultured majority of the sponge microbiota.  For this approach large clone metagenomic libraries utilising customized vectors are being used to enable functional expression in several host strains.  Due to the large numbers of clones required for this approach a robotic colony picking and arraying system are being employed.  Positive clones are being analysed by subcloning, expression, and DNA sequence analysis.

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Marlies Mooij [email:- m.vanderwal-mooij@ucc.ie]

Cell Signalling in Marine Microorganisms-Biofilms-Biofouling

Cell signalling systems and the various genes under its control are crucial for bacterial virulence. As such, these signalling systems could serve as novel targets in the development of new therapeutic agents.  Many bacteria use the cell density dependent cell to cell signalling system, quorum sensing (QS) to coordinate their virulence, including, motility, the formation of biofilms and the expression of drug resistance efflux pumps. Therefore, quorum sensing is a promising target for antimicrobial drugs. This work is focusing on Pseudomonas aeruginosa, as a model system. P. aeruginosa is a ubiquitous environmental bacterium that is among the top three opportunistic human pathogens, and has previously been used as a model organism for studying cell signalling. Reporter assays are being used to identify novel QS and biofilm inhibitors from marine sponges and from marine sponge associated microorganisms, which are a rich source for structurally novel biologically active compounds. Functional metagenomic based approaches are also being employed.  In addition a recently established zebrafish facility within the BIOMERIT Research Centre at the Microbiology Department at University College Cork, is employing zebrafish embryos as an in vivo biological model, to screen for drug interventions on bacterial infections, as well as for effects that bioactive compounds may have on the development of the zebrafish embryo.

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 Research Programmes for PhD Students


Burkhardt Flemer

 

Burkhardt Flemer [email:- bflemer@gmail.com]

Project Title

Antimicrobial activities from marine sponges and sponge derived microbes

Supervision Team

Prof Alan Dobson, Prof Fergal O’Gara, Dr John Morrissey and Dr Jonathan Kennedy

Collaborating Scientist

Dr. Ruan Edrada-Ebel. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde

Overview

Initial work in the Cork group, using both culture dependent and independent techniques has  established that the sponge Haliclona simulans has a high microbial diversity and that many of the cultured microbes produce antimicrobial activities.  Several of these activities have been shown to have broad antimicrobial activity and can be extracted from the culture broth by organic extraction techniques.  Activities from these sponge derived microbes will be further characterised during the course of this project to determine the chemical structure and potential novelty of the active components.  In addition a diverse range of additional sponge specimens will be collected during the course of this project and extracts of these will also be screened for the presence of antimicrobial substances.  Additional microbial species isolated from these newly collected sponges will also be screened for the presence of additional antimicrobial activities, and activities will be analysed as above. Finally sponge metagenomic libraries will be constructed and screened for antibiotic and other activities, while selected strains or metagenomic clones will be further analysed to determine the genetics, biochemisty and regulation of antibiotic biosynthesis.

Overall project aims

The project aims to screen a range of extracts from marine sponges for antimicrobial activities and also isolate a wide range of microbes from marine sponge samples.  In addition a range of sponge-derived microbes will be screened for antimicrobial activities, together with sponge metagenomic libraries. Finally active components from either bacterial samples or metagenomic libraries will be extracted and identified.

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Stephen Jackson

Stephen Jackson [email:- sjackson@ucc.ie]

Project Title

Microbial diversity in marine sponges from Irish waters

Supervision Team

Prof Alan Dobson, Dr John Morrissey, Prof Fergal O’Gara and Dr Jonathan Kennedy

Overview

This project will focus on the microbial ecology of a range of sponge specimens (including Cliona celata, Polymastia boletiformis, Suberites carnosus, Axinella dissimilis, Tethya citrina, Raspailia ramosa and Stelligera stuposa.  The project will determine what microbes are present in the different sponge samples and compare the microbial content both within and between sponge species, using a range of molecular and microbiological techniques (e.g. RFLP, DGGE, DNA sequencing /Pyrosequencing and microscopic techniques).  Culture dependent techniques will be employed to assess the culturable microbial diversity of a subset of these sponge species, while group specific probes will be developed to determine the location of specific microbial groups within the sponge tissues, using fluorescent in-situ hybridization. An additional part of the project will be to construct metagenomic libraries and to functionally screen these libraries for the presence of activities of biotechnological relevance (e.g. enzymes and antibiotics), adapting the screens and methods used to the known microbial diversity of the sponge.

Overall project aims

This project aims to assess the full microbial diversity of a number of marine sponges, but in particular to focus on Raspailia ramosa and Stelligera stuposa and to elucidate the bacterial community profiles associated with these two marine sponges. Attempts will be made to identify microbial groups that are specific to particular sponge species and those which are found in a number of sponge species. In addition microbes will be cultured from these sponges using novel isolation methods to try to access previously uncultured sponge microbes. Finally metagenomic techniques will be employed to exploit the microbial metabolic diversity of the sponge microbiota.

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Lekha Menon Margassery

Lekha Menon Margassery [email: lekha@student.ucc.ie]

Project Title

Development and validation of high-throughput screens to identify novel inhibitors of pathogenic yeast, from marine sponge-associated microbes.

Supervision team

Dr John Morrissey, Prof. Alan Dobson, Prof. Fergal O’Gara, Dr Jonathan Kennedy

Overview

Preliminary work has already established that cultured marine-associated microbes secrete metabolites that are inhibitory to yeast growth. Initially the ability of various cultured microbes to inhibit different yeasts will be tested and a bank of species identified from which molecules with anti-fungal but not anti-bacterial activity can be identified.  Using a variety of reporter strains including gene fusions and physiological probes; the target(s) or mode of action of these molecules will then be assessed.  This reporter collection to assess whether particular target genes are activated as well as strains mutated or overexpressing candidate target proteins.  Physiological reporters include (fluorescent) molecular probes to measure mitochondrial function, membrane integrity and oxidative stress.

A high-throughput assay will be developed to enable screening of large numbers of extracts from strains or from metagenomic libraries.  This will use gene reporters that respond to activated calcineurin and the system will be designed to allow robotic manipulation and microtitre plate analysis. Inhibitors that are identified will be assessed in secondary screens to assess specificity (target and organism).Finally novel inhibitors will be subjected to molecular chemical analysis, while molecular analysis will be performed to determine what genes encode the molecule.

Overall project aims

This project aims to establish the cellular targets of antifungal extracts of sponge-associated bacteria using a Saccharomyces cerevisiae system and to develop a scaleable high-throughput assay to screen for inhibitors of the calcium-activated phosphatase, calcineurin, in yeast. It also aims to screen extracts of of marine sponges-associated microbes and metagenomic libraries from such microbes to identify novel calcineurin inhibitors and to biologically characterise calcineurin inhibitors to determine specificity. Finally it also aims to determine the chemical nature of these novel inhibitory moleucles.

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Julianne Megaw

Julianne Megaw  [email:- j.megaw@qub.ac.uk]

Project Title

Limits of cell function & stress responses of marine microbes: biotechnological applications

Supervision Team

Prof. Brendan Gilmore

Overview

This project will focus on marine microbes, especially extremophilic species, from diverse marine habitats (including those not previously studied). A culture collection will be made of these species, and studies performed on their stress tolerance & responses alongside model species in order to select metabolically/ ecophysiologically novel strains. The stress metabolism of these strains will be manipulated in order to characterise their biochemical/ phenotypic potential. This information will then be employed to ‘mine’ their global cellular responses for pharmaceutically & biotechnologically useful products.

Overall Project Aims

This project aims to sample and screen geographically and physicochemically diverse marine environments (slitch, and ancient salt deposits) for stress-tolerant microbes using diverse culturing strategies (chao- and kosmotropic media, low water activity etc); to isolate, screen for and identify exceptionally stress-tolerant strains via comparisons with know marine extremophiles and to make a culture collection of new strains. The stress-tolerance phenotypes of the most stress-tolerant strains will be characterised over a matrix of diverse stress parameters, including biotech-relevant stress parameters. To manipulate the stress metabolism of some of these strains in order to characterise their biochemical/ phenotypic potential and use this information to ‘mine’ their global cellular responses for pharmaceutically & biotechnologically useful products. Screens will be carried out for antimicrobial activity using a range of model pathogens, for antibiofilm activity and for other pharmaceutically relevant activities.

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Robert Phelan [email:- 105406340@umail.ucc.ie]

Project Title

Characterisation of sporeforming bacteria and yeasts from Irish marine sponges

Supervision team

Dr Teresa Barbosa, Dr John Morrissey, Prof. Fergal O’ Gara, Prof. Alan Dobson

Overview

Most studies of culturable sponge-associated microbes have focused on vegetative bacterial cells with little attention being paid to spore-forming bacteria, such as Bacillus spp. Sporeformers, like Bacillus sp., offer numerous advantages over other marine bacteria in that, tools for their genetic manipulation already exist and they have a track record of bioactive compound production. It has been reported that approximately 5% of the genome of B.subtilis is dedicated to production of antibiotics. The general attitude towards Bacillus sp isolated from marine habitats in the past was that they were present as contaminants. Nowadays it is believed that the marine environment may represent a new habitat for Bacillus sp. There is very little known about the diversity and specific properties of marine sporeformers and how they may differ from their terrestrial counterparts. Information on this group of microorganisms will provide us with the ability to characterise these marine bacteria and ultimately provide us with the tools required to exploit them as a source of novel pharmaceutical and biotechnological compounds. This project will also focus on marine yeasts, which comprise another group of microbes that have not received much attention. This classification cover diverse unicellular fungi, some of which may prove valuable as sources of enzymes or bioactive molecules.

Overall project aims

This project aims to characterise the sporeforming population with the marine sponge H. simulans and to determine if the marine Bacillus sp. differ in comparison to their soil counterparts. To screen these marine bacilli for antibiotic production and to purify and characterise any antimicrobial agents produced by these bacteria and yeast. To identify Quorum Sensing inhibiting compounds from marine sponge associated bacteria and finally to assess the diversity and biotechnological potential of marine-sponge associated yeasts.