Transcript Diversity and similarity analysis of microbial communities

Work package 3
Analysis of the species diversity,
community structure and phylogeny of
microorganisms.
Laboratory of Microbial Ecology
CEES/RUG
Objectives
• Evaluate microbial communities in 4 deep sea
hypersaline anoxic basins (DHABs)
• Evaluate similarities between DHABs and
between different positions in one DHAB
• Link previously published geochemical data
with these results
Brine interface water
Filter
DNA-isolation
Bacterial 16S
rDNA PCR
Archaeal 16S
rDNA PCR
Cloning PCRproduct
Cloning PCRproduct
Sequencing
700 bases
Sequencing
700 bases
Bacteria
Brine
Interface
Seawater
Atalante
Bannock
Discovery
Urania
Atalante
Bannock
Discovery
Urania
Bannock
Discovery
Sequences
90
90
90
90
90
90
90
90
90
90
OTUa
40
41
48
16
28
27
36
25
39
57
Shannon
3.26
3.20
3.51
2.17
2.90
2.72
3.27
2.78
3.42
3.88
Archaea
Brine
Interface
Seawater
Atalante
Bannock
Discovery
Urania
Atalante
Bannock
Discovery
Urania
Bannock
Discovery
Sequences
90
90
90
90
90
90
90
90
90
90
OTU
11
10
10
5
15
14
15
16
17
12
Shannon
1.60
1.49
1.55
0.61
2.01
1.83
1.77
1.44
2.30
1.87
PCoA of Morisita’s Index for Bacterial OTU-level
Coordinate 2
17.9% of Variance
0.7
DB
0.6
0.5
0.4
0.3
0.2
DI
0.1
0 UI
-0.1
BI
AI
-0.2
-0.3
AB
UB
BB
D3300
B3000
-0.3 -0.2 -0.1
0
0.1 0.2 0.3 0.4 0.5 0.6
Coordinate 1
19.9% of Variance
PCoA of Morisita’s Index for Archaeal OTU-level
0.8
D3300
Coordinate 2
18.2% of Variance
0.6
0.4
0.2
B3000
AI
0
-0.2
-0.4
AB BB
UB
DI
DB
BI
UI
-0.4 -0.3
-0.2
-0.1
0
0.1
Coordinate 1
51.5% of Variance
0.2
0.3
0.4
Cluster Analysis (UPMGA) of Morisita’s Index for OTUlevel
1.0
UB
BB
AB
DB
0.8
0.6
0.4
0.2
Bacterial Brine+Interface
Archaeal Brine
Archaeal Interface
PCoA of Morisita’s Index for Bacterial Phylogenetic
Group-level
0.4
0.2
0.1
Coordinate 2
23.3% of Variance
0.3
DI
DB
AB
UB
BB
0
-0.1
-0.2
UI
BI
-0.3
-0.4
-0.5
AI
B3000
D3300
-0.4 -0.3 -0.2 -0.1
0 0.1 0.2 0.3 0.4 0.5
Coordinate 1
45.3% of Variance
PCoA of Morisita’s Index for Archaeal Phylogenetic
Group-level
DB
Coordinate 2
7.6% of Variance
0.8
0.6
0.4
0.2
DI
D3300
0 B3000 BI
UI
-0.2
-0.4
-0.3
UB
AI
BB
AB
-0.2
-0.1
0
0.1
Coordinate 1
88.0% of Variance
0.2
0.3
Principal Component Analysis
• The two seawater samples always group
together
• Discovery brine and Discovery Interface always
group together
• L’Atalante, Urania and Bannock brines group
together except for Bannock Bacterial
• L’Atalante, Urania and Bannock interface
group together except for L’Atalante Archaea
• In most cases, Discovery samples group
together with other brine samples
Cluster analysis
• Discovery is most different from the
other DHABs for the bacterial and
archaeal brine and the bacterial interface
samples.
• Next to Discovery, Urania is most
different from the other DHABs
• Two clusters were observed for the
archaeal interface samples
Relative Distribution of Phylogenetic Bacterial Groups
50.0
AB
40.0
BB
35.0
DB
30.0
UB
AI
25.0
BI
20.0
DI
15.0
UI
10.0
B3000
D3300
5.0
KB1
gammaproteo
new/unknown
5
epsilon
proteo
Bacteroidetes
0.0
delta-proteo
Percentage (%)
45.0
Relative Distribution of Phylogenetic Archaeal Groups
100
90
Percentage (%)
80
AB
BB
70
DB
60
UB
AI
50
BI
DI
40
UI
30
B3000
D3300
20
10
0
Marine Group I
MSBL1
Halobacteriales
Metabolic Routes
- High number of δ-proteobacteria
 Sulfate reduction Important
 H2, organic matter or anaerobic CH4 oxid.
No archaea related to anaerobic CH4 oxid.
δ-13C CO2 values are not extremely low
 H2 and organic matter most important for
SO4-reduction
Metabolic Routes
• ε- and γ-proteobacteria were mainly related to
sulfide oxidizing bacteria.
• These sequences were most numerous in
Urania
• Sulfide oxidation plays an important role in the
interface samples
• High number of Bacteroidetes shows that
heterotrophic bacteria play important role
Archaea
• Archaea related to Marine group I and a
new cluster MSBL1
• At present unknown what the function is
of these archaea
• Discovery high number of Halorhabdus
utahensis
• This bacterium adapted to high
concentrations of Mg2+
Conclusions
• Interface samples different from brine samples.
The sharp increase in salinity, sulfide and
decrease in O2 are very important factors for
the establishment of a microbial community.
• Discovery different. MgCl2 is thus important
factor in establishment of microbial community
• Next to Discovery, Urania differs. Sulfide
concentration is an important factor as well.
Conclusions
• Microbial routes present in the sulfur cycle
have an important function in the DHABs
• Most of the archaeal sequences form a new
branch in the archaea tree. This cluster is
named MSBL1
• The function of almost all archaea as well for a
great number of bacteria is at present unknown
Bannock interface samples cruise II
33.3% of Variance
1B 1A
4A
4B
3A
3B
2B
2A
45.5% of Variance
Future Plans
• A complete phylogenetic analysis with
GenBank database of the Bannock
interface samples
• Study temporal variation by comparing
sequences from brines of cruise I, II (and
III?)
• Study diversity of several functional
genes or specific groups with specific 16S
rDNA primers