1. Environmental Biochemistry University of Oldenburg Fremantle, 2013
Georg Steinert1, Susanna Whitfield2, Mike W. Taylor3, Peter J. Schupp1 Institute for the Chemistry and Biology of the Marine Environment, University of Oldenburg, Germany1 Marine Laboratory, University of Guam, Mangilao, Guam2 Centre for Microbial Innovation, School of Biological Sciences, University of Auckland, Auckland, New Zealand3
Environmental Biochemistry
University of Oldenburg
Fremantle, 2013
Phylogenetic analysis
of bacteria isolated from sponges
using a diffusion-growth-chamber

2. The premise and the problem
Marine sponges contain dense and diverse microbial communities
up to 35% of sponge biomass
renowned sources of bioactive metabolites
Natural products span a broad spectrum for applications
anti-bacterial
anti-cancer
anti-viral
anti-fouling
Potential of sponge-derived compounds is not fully realized
“supply-issue”
uncultivable microbes (0.04 – 11%, species specific)
novel microbes & novel compounds

3. Diffusion-growth-chambers
Diffusion-growth-chamber (DGC)
in vivo inoculation in Rhabdastrella globostellata (Guam, USA)
4 generations & 4 media types (M8, MB1:10, M1, M1 with sponge extract)
parallel one direct cultivation generation
16S rRNA genome Sanger sequencing
Aims
can we access the cultivable microbial R. globostellata community?
can we cultivate sponge-specific bacteria?
can we cultivate previously uncultivable bacteria (OTUs < 97% similarity to cultured ones)?

4. Results – bacterial community
Identification of cultivated bacteria via ARB 16S rRNA analysis
Congruent to cultivable R. globostellata community accessed by Lafi et al. 2005

5. Bacteroidetes Sponge-specific-cluster
sponge-specific ARB database by Simister et al. 2012
RAxML
1000 replicates
fast bootstrap
Maximum likelihood
Neighbor joining
Maximum parsimony

6. Gammaproteobacteria Sponge/Coral specific cluster
sponge-specific ARB database by Simister et al. 2012
RAxML
1000 replicates
fast bootstrap
Maximum likelihood
Neighbor joining
Maximum parsimony

7. Environmental Biochemistry University of Oldenburg Fremantle, 2013
Take it home in a nutshell
Environmental Biochemistry
University of Oldenburg
Fremantle, 2013
in vivo inoculation and cultivation is possible via DGC technique
Cultivable bacterial phyla are congruent to recent cultivation approaches (Lafi et al. 2005):
Actinobacteria
Bacteroidetes
Firmicutes
Proteobacteria (Alpha & Gamma)
15 novel strains and/or 3 OTUs (OTUs < 97% similarity to cultured ones)
No sponge-specific-cluster from DGCs
most bacterial species and species-level OTUs are limited to single host-species (see Schmitt et al. 2012)
SCC approach good for core and variable community, but what about the host species-specific community?
Outlook
more different media types, e.g. spongin (see Sipkema et al. 2011)
greater variety of sponge species
high throughput sequencing

8. UGO Thank you for your attention And many thanks to: Mike Taylor Lab
Carsten Thoms
Guam Marine Lab Crew
Institute of the Chemistry and Biology of the Marine Environment
Institute and cooperation partner:
Conference scholarships:
German Academic Exchange Service
UGO
Universitätsgesellschaft Oldenburg

9. Sponge specific 16S rRNA sequence clusters
Definition by Hentschel et al. (2002):
A group of at least 3 sequences
recovered from different sponge species and/or different geographic locations
more closely related to each other than any other sequence from non-sponge sources
cluster together independent of applied phylogenetic method
see further work in Taylor et al and Simister et al. 2012
Maximum likelihood
Neighbor joining
Maximum parsimony

10. Environmental Biochemistry University of Oldenburg Fremantle, 2013
Experimental setup
Environmental Biochemistry
University of Oldenburg
Fremantle, 2013

11. Sponge specific 16S rRNA sequence clusters
not a sponge-specific-cluster A B C
Maximum Likelihood
Neighbor Joining
Maximum Parsimony

12. Sponge specific 16S rRNA sequence clustersB C
Maximum Likelihood
Neighbor Joining
Maximum Parsimony
sponge-specific-cluster

13. Bacterial community results
Environmental Biochemistry
University of Oldenburg
Fremantle, 2013
Table 2. Number of OTUs with 0.03 distance cutoff level, number of novel 0.03 UTOs with <97% similarity to cultivated bacteria, number of novel full sequences with <97% similarity to cultivated bacteria