BIODEEP (EVK3-2000-00042) First year Scientific Report
WP3 - ANNEX
Contribution of partner 6: UESSEX, Department of Biological Sciences, University of Essex,
Central Campus, Wivenhoe Park, Colchester. CO4 3SQ. U.K.
Scientists involved: Andrea M. Sass and Terry J. McGenity
3.1 Objectives
To obtain an overview of biodiversity in the deep anoxic hypersaline basins, the overlying oxic
seawater and the interface, using terminal restriction fragment length polymorphism (t-RFLP).
3.2 Methodology and Scientific Achievements
Methods
The water samples were filtered through Durapore membrane filters with 0.1 µm pore size. The
filters were preserved and the microbal cells lysed in guanidium thiocyanate solution (5 M
guanidium thiocyanate, 0.5 M TRIS pH 6.4, 0.02 M EDTA pH 8.0 and 1.3% (w/v) triton X100). Recovery of DNA was performed by binding to glassmilk (Bio101), washing with 70 %
(v/v) ethanol and subsequent elution from the glassmilk with 10 mM TRIS pH 8.
Sediment samples were directly mixed with guanidium thiocyanate solution; the suspension was
then centrifuged and the DNA in the guanidium thiocyanate-containing supernatant was
recovered as described above.
Bacterial 16S rDNA was amplified from the extracted DNA. Each PCR contained, per reaction:
water, 34.5 l; 10 buffer (Qiagen), 5 l; dNTPS (2 mM), 5 l; forward and reverse primers (10
M), 2 l; Taq DNA polymerase (Qiagen; 5 units l-1), 0.5 l; DNA, 1 l. Reaction conditions
were: initial denaturation at 94 C for 2 min, followed by 30 cycles of denaturation (94 C for 1
min), annealing (55 C for 1 min), and extension (72 C for 2 min), with a final extension time
of 10 min. The forward primer was FAM-63F (5-CAG GCC TAA CAC ATG CAA GTC-3)
and the reverse primer was HEX-1389R (5-ACG GGC GGT GTG TAC AAG-3), labelled at
the 5 end with the fluorescent dyes, 6-FAM and HEX (Applied Biosystems) respectively.
PCR products were cleaned using QIAquick columns (Qiagen), and 10 l was mixed with 2 l
of restriction enzyme (CfoI and AluI from Boehringer Mannheim, in separate incubations, both
at 10 units l-1) and 1.3 l of 10 buffer, and incubated at 37 C for 3 h. One l of the digested
product was mixed with 12 l of deionised formamide and 0.5 l of the ROX-labelled GS500
internal size standards (Applied Biosystems), denatured by heating to 95 C for 5 minutes and
separated by capillary electrophoresis using POP4 polymer (ABI) on an ABI 310 automated
DNA sequencer.
GeneScan software (Applied Biosystems) was used to analyse the t-RFLP profiles. Band sizes
and peak areas were determined and then normalised by dividing the total peak area for each
sample by the mean total peak area of all samples. The resulting value was used as the minimum
peak hight allowed, and peaks were detected again using GeneScan software. This approach
corrected for differences in the amount of DNA loaded. A table of peak size (base pairs) against
peak areas (relative to the maximum peak) in each sample was used to calculate the Euclidean
distance (MVSP software) between each pair of samples. This analysis resulted in a matrix of
pairwise distances, which was converted into a tree using the unweighted pair group method
using arithmetic averages (MVSP software).
BIODEEP (EVK3-2000-00042) First year Scientific Report
Results
To date, the amplification of Bacterial 16S rDNA has been successful using DNA from oxic
seawater samples, interfaces and brines of L’Atalante, Urania and Bannock basins (Table 1).
The DNA extraction from interface and brine samples of the Discovery basin yielded rather low
molecular weight DNA or RNA only. The first attempts of DNA extraction from the sediment
samples were unsuccessful.
Table 1: Sample details and allocated codes
Code for water Location of sample
sample
A
B
C
D
DI
DB
UI
UB
AI
AB
BI
BB
16S rDNA
amplification
Seawater near Discovery, CTD 1, 2500 m depth
Seawater near Discovery, CTD 2, 3500 m depth
Seawater near Discovery, CTD 4, 3300 m depth
Seawater near Bannock, CTD 7, 3000 m depth
Interface of Discovery basin
Brine of Discovery basin
Interface of Urania basin
Brine of Urania basin
Interface of L’Atalante basin
Brine of L’Atalante basin
Interface of Bannock basin
Brine of Bannock basin
+
+
+
+
+
+
+
+
+
+
The analysis of the restriction digestion of the PCR products revealed a relatively high similarity
between the samples from oxic seawater (A, C, D), the two samples from the same site were
most similar (A, C). Relatively high similarity was also observed between the deepest oxic
seawater sample (B) and the Urania interface sample (Fig. 1), and between the samples from
l’Atalante and Bannock basin brines and l’Atalante basin interface. Urania basin brine has the
most different microbial community on the basis of t-RFLP analysis.
UPGMA
1.5
1.25
1
0.75
UB
BI
AI
BB
AB
UI
B
D
C
A
0.5
0.25
0
Euclidean distance
Fig. 1: Clustering of t-RFLP profiles, data from
restriction digestion with Alu I and Cfo I combined
BIODEEP (EVK3-2000-00042) First year Scientific Report
3.4 Discussion and Conclusions
The sampling techniques resulted in the interface samples being, to differing degrees, a mixture
of oxic seawater, interface and the respective brines. This explains the relationship between the
community fingerprints derived from interfaces and from brines, or between the fingerprint
derived from Urania interface and the seawater sample from 3500 m depth. Many terminal
restriction fragments from interface samples were also found in samples from the respective
brine or from oxic seawater. However, terminal restriction fragments unique to samples from the
interfaces were also seen. This may be the result of a shift in abundance of organisms due to
accumulation of microorganisms from the oxic seawater at the density barrier of the interface, or
to the presence of microorganisms specifically adapted to the interface environment with its
steep chemical gradients.
The fingerprints from brine samples were less similar to each other than the oxic seawater
samples A, C, and D from different depths and locations. The brines therefore have a microbial
community unique to the respective location. The communities in the brines from l’Atalante and
Bannock basins are more similar to each other than to the community inhabiting the Urania
basin brine. This is reflected in the chemical composition of the brines which is rather similar for
l’Atalante and Bannock basins, whereas the Urania basin brine is characterised by uniquely high
sulphide and methane concentrations.
3.5 Plan and Objectives for the Next Period
Different DNA extraction protocols will be applied to the sediment samples to obtain microbial
community profiles. These community profiles will be compared with those from anoxic
enrichment cultures of sediment samples in order to determine whether enriched
microorganisms are abundant in the sediments.
The Archaeal community in the seawater, brine and interface samples will be profiled using tRFLP.