University of Essex BIODEEP-WP3 Analysis of species diversity, community structures and phylogeny of microorganisms and meiofauna in the Mediterranean deep-sea hypersaline anoxic basins (DHAB) Andrea Sass, Terry McGenity

Genes from different microorganisms Methods:  Cell preservation and lysis  DNA recovery and cleaning  Amplification of 16S rRNA gene with eubacterial and archaebacterial primers, labelled with fluorescent dyes  Digestion with restriction enzymes  Separation of DNA fragments  Detection of label  Alignment of fragments  Cluster analysis University of Essex Total DNA Amplification of 16S rRNA gene Gene fragments Digestion with restriction enzyme

oxic water near Discovery basin 2500 m depth oxic water near Discovery basin 3300 m depth oxic water near Bannock basin 3000 m depth Electropherograms of three samples of oxic water from different depths (restriction enzyme: Alu I), size of ordinate adjusted according to amount of DNA in the sample A C D Fragment size (base pairs) Peak height (rel. fluorescence units)

Samples included in analysis: Interface brine and brine body from l’Atalante, Urania and Bannock basins University of Essex Other samples: Discovery brine and interface: DNA extracted but no amplification possible Sediments: no DNA could be extracted A near Discovery basin, CTD 1, 2500 m depth B near Discovery basin, CTD 2, 3500 m depth C near Discovery basin, CTD 4, 3300 m depth D near Bannock basin, CTD 7, 3000 m depth Oxic water:

DIBIAIUIBSAS bp 2072 bp 600 bp Agarose gel of DNA extracts

Clustering of t-RFLP fingerprints, data from restriction digestion with Alu I and Cfo I combined Euclidean distance Urania basin brine Bannock basin interface l‘Atalante basin interface Bannock basin brine l‘Atalante basin brine Urania basin interface B, oxic water, 3500 m depth D, oxic water, 3000 m depth C, oxic water, 3300 m depth A, oxic water, 2500 m depth University of Essex

Electropherograms of the brines of three basins (restriction enzyme: Alu I), size of ordinate adjusted according to amount of DNA in the sample Fragment size (base pairs) Peak height (rel. fluorescence units) l‘Atalante basin brine Bannock basin brine Urania basin brine

Fragment size (base pairs) Peak height (rel. fluorescence units) Electropherograms of the interfaces of three basins (restriction enzyme: Alu I), size of ordinate adjusted according to amount of DNA in the sample L‘Atalante basin interface Bannock basin interface Urania basin interface

Fragment size (base pairs) Peak height (rel. fluorescence units) Oxic water, B near Discovery basin 3500 m depth Urania basin interface L‘Atalante basin interface L‘Atalante basin brine Electropherograms of the brines of three basins (restriction enzyme: Alu I), size of ordinate adjusted according to amount of DNA in the sample

University of Essex  The basin brines contain unique microbial communities  The differences in community structure profiles of the brines possibly reflect the difference in their chemical composition  A microbial community unique to the interfaces may exist Conclusions:

Future work: Comparison of t-RFLP patterns of sequences from isolates and clones with the patterns from the total community Evaluation of the abundance of isolates and clones in situ Further attempts to obtain DNA from sediments