Sulfate reducing prokaryotes in the Eastern Mediterranean

A functional genomics approach

• Sulfate reduction:– SO4

2- + 8H+ +8e- S2- + 4H2O

– Electron donors• Organic matter (lactate, acetate, ethanol, etc)

• H2

• CH4

– Important in anoxic marine ecosystems but occurs in other ecosystems as well.

Sulfate reducing prokaryotes

• Dissimilatory sulfite reductase (DSR)– Enzyme involved in sulfate reduction– Catalysis following reaction:

– The gene encoding for enzyme contains conservative and variable sites

– Therefore a good gene to study diversity of sulfate reducing prokaryotes in the environment

Deep hypersaline brines

• Eastern Mediterranean contains hypersaline brines which are located at the deep-sea.

• These brines are characterized by high salinity (up to 30% salt), high pressure (up to 350 bar) absence of oxygen and relatively high concentrations of sulfate and sulfide.

• 16S rDNA sequence analysis revealed many sequences related to δ-proteobacteria.

• Sulfate reduction rates ranged from 8 to 80 µmol H2S day-1 in the different brines.

• Conclusion:– Sulfate reduction occurs as a metabolic

process in deep hypersaline brines

Deep hypersaline brines

Objectives

• What is the similarity of SRP communities between different sampling sites

• Is their similarity between DSR sequence analysis and 16S rDNA sequence analysis.

• What is the community structure of sulfate reducing prokaryotes (SRP)?

Mat & Meth

• Study sites:– L’Atalante brine and interface– Urania brine and interface– Eastern Mediterranean deep-sea sediment three

layers

• α- and β-subunit of DSR gene amplified• 700 bp of α-subunit were sequenced• Amino acid alignments were created and trees

were constructed using these alignments

Diversity and SimilarityDiversity Atalante brine Atalante interface Urania brine Urania interface Sed1 Sed2 Sed3

DSR δ-16S DSR δ-16S DSR δ-16S DSR δ-16S DSR DSR DSR

Sequences 100 18 100 6 100 28 100 16 22 21 10

Shannon index 1.5 2.0 0.99 1.3 0.28 0.71 1.67 1.2 1.3 2.0 1.6

Similarity Atalante brine Atalante interface Urania brine Urania interface Sed1 Sed2 Sed3

DSR δ-16S DSR δ-16S DSR δ-16S DSR δ-16S DSR DSR DSR

AB DSR/16S 1 1 0.26 0.00 0.00 0.22 0.00 0.05 0.00 0.00 0.00

AI DSR/16S 1 1 0.00 0.00 0.00 0.03 0.00 0.00 0.00

UB DSR/16S 1 1 0.00 0.31 0.00 0.00 0.00

UI DSR/16S 1 1 0.00 0.00 0.00

Sed1 DSR/16S 1 0.00 0.00

Sed2 DSR/16S 1 0.00

Sed3 DSR/16S 1

Nearest relatives DSR-proteinOTU Nearest relative similarity

46 AB 56 Desulfohalobium retbaense 83.4%

11 AB 29 Desulfohalobium retbaense 83%

11 AB 90 Desulfobacter vibrioformis 85%

23 AB 95 uncultured deep-sea hydrothermal vent 1 77%

75 AI 36 Desulfobacter vibrioformis 86%

6 AI 18 Desulfobacter vibrioformis 86%

5 AI 60 unidentified bacterium 87%

8 AI 37 uncultured deep-sea hydrothermal vent 1 76%

94 UB 28 uncultured deep-sea hydrothermal vent 1 76%

11 UI 91 Desulfobacter vibrioformis 85%

5 UI 15 Desulfobacterium oleovorans 82%

5 UI 7 Desulfobacterium oleovorans 85%

6 UI 43 uncultured bacterium 1 84%

11 UI 14 unidentified bacterium 92%

52 UI 75 unidentified bacterium 93%

13 Sed1 01 uncultured bacterium 2 84%

7 Sed2 09 uncultured Guaymas Basin 64%

3 Sed3 01 uncultured deep-sea hydrothermal vent 1 84%

Phylogenetic tree

DSRa-protein

Phylogenetic tree

δ-16S rDNA

δ-Proteobacterial family distribution

0

10

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30

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50

60

70

80

90

100

DSR 16S DSR 16S DSR 16S DSR 16S

AB AI UI UB

Pe

rce

nta

ge

of

clo

ne

s

Dulfohalobiaceae

Desulfobacteriaceae

Desulfovibrionaceae

Desulfobulbaceae

Origin of sulfate reduction

D esu lfobo tu lu s sa po vo ra ns

D esu lfoha lob ium re tba en se

D esu lfoba cu la to luo lica D esu lfovibrio fru cto sovo rans

D esu lfosarc ina va riab ilis D esu lfo tom a cu lum ku znetso vii

A rcha eog lob us pro fu ndu s A rcha eog lob us fu lg id us

D esu lfo tom a cu lum rum in is T h erm odesu lfo vib rio is la nd icus

Percentage of clones without insertion

0

10

20

30

40

50

60

70

80

90

100

AB AI UB UI sed1 sed2 sed3

pe

rce

nta

ge

of

clo

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s

Conclusions/Discussion• All sites sampled showed diverse sulfate

reducing prokaryotic communities except Urania brine.

• The low diversity in Urania brine has been observed with total community structure as well.

• Similarity of DSRa sequences between sites is very low thus each site studied had a unique sulfate reducing community.

• There are some differences between site similarity of DSRa and δ-16S rDNA. Can be related to OTU cut-off value or that not all DSRa sequences are from δ-proteobacteria

Conclusions/Discussion• The obtained DSR-sequences show low

similarity with GenBank sequences and represent yet-unknown DSRa genes from sulfate reducing prokaryotes.

• The DSRa and 16S rDNA tree topology and family distribution were similar for AI, UB and AB.

• This was not true for UI. UI DSRa sequences distantly related to Desulfotomaculum but no 16S rDNA sequences related to that cluster.

Conclusions/Discussion

• This can be caused by– 1. These DSRa sequences are related to the

δ-16S rDNA sequences but this cannot be seen because tree topologies are non congruent

– 2. 16S rDNA sequences of UI related to unknown or candidate division clusters, from which metabolic capacities are unknown, are from prokaryotes with sulfate reducing capabilities.

Conclusions/Discussion• Allmost all DSRa sequences from deep-sea brines

and interfaces contain an insertion in α-subunit.– This might indicate that sequences are from non-

thermophilic sulfate reducing prokaryotes

• Most DSRa sequences from intermediate layer sediment miss this insertion.– This might indicate that sequences are thermophilic

sulfate reducing prokaryotes. This agrees with the thermogenic history. Why these sequences only occur at the intermediate layer is presently unknown.