1

Nullarbor Cave Slime:

Looking at life in the dark

2

Location of sampled caves

3

Beneath the Nullarbor…

Entrance to Warbla cave, Western Australia- photo Liz Rogers

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Cave passage flooded with saline water

Photo - Liz Rogers

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Sampling procedure

Photo - Steve Trewavas

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Microbial ‘Slime’ Communities

SEM of slime curtain material

(from Holmes et al 2001)

Calcite Crystal

Filaments Microbial Cells

Photo- Peter Rogers

7

0 10000 20000 30000 40000 50000 60000 70000

0

10

20

30

40

50

60

70

80

90

100

Contig Size vs Coverage

Contig Size

Cont

ig C

ove

rage

(nu

mber

of re

ads)

Metagenome sequencing of Weebubbie cave

community

• Environmental DNA from Weebubbie cave was sequenced

with 454 GS-FLX (Ramaciotti centre)

• Obtained 548 365 reads, average length 581bp

• 279 393 (~51%) assembled into contigs

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Inferred taxonomic composition

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Nitrosopumilus

maritimus SCM1

Weebubbie

Metagenomic Contigs

Alignment of Weebubbie contigs and Nitrosopumilus

maritimus SCM1 genome

Almost all large contigs showed significant similarity to the

Nitrosopumilus maritimus SCM1 genome

10

Nitrosopumilus

maritimus SCM1

Weebubbie

Metagenomic Contigs

Alignment of Weebubbie contigs and Nitrosopumilus

maritimus SCM1 genome

Nitrosopumilus

maritimus SCM1

Weebubbie

Metagenomic Contigs

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Phylogenetic tree- Weebubbie archaeal 16S rRNA

gene T

hau

march

aeo

ta

Cren

arch

aeo

ta

Eu

ryarch

aeo

ta

12

Functional comparison of Weebubbie metagenome

with other aquatic metagenomes

Weebubbie Cave Metagenome ♦

Chesapeake Bay Estuary, U.S.A. ■

South Australia Groundwater Aquifer ●

Coastal Caribbean Sea ■

Isabella Island Mangroves, Galapagos Island ■

Deep Marmara Sea (1000 m) ■

Cell Division and Cell Cycle

Nitrogen Metabolism

Potassium Metabolism Photosynthesis

Iron Acquisition and Metabolism

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

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Ammonia

Hydroxylamine

Nitric Oxide Dinitrogen

Oxide Nitrogen

Nitrate

Nitrite

Bacteria Archaea

Ammonia

Monooxygenase

(108) EC 1.13.12.-

Hydroxylamine

Oxidase (8)

EC 1.7.3.4

Nitrate Reductase

NADH (42)

EC 1.7.1.1

Nitrite Reductase

NAD(P)H (42)

EC 1.7.1.4

Ferredoxin Nitrite

Reductase

(74) EC 1.7.7.1

Nitric Oxide

Reductase (10)

EC 1.7.2.5

Nitrous Oxide

Reductase (28)

EC 1.7.2.4

Nitrate

Reductase (314)

EC 1.7.2.1

Nitrite Reductase

NO Forming (218)

EC 1.7.2.1

Nitrate

Oxidoreductase

(79)

Bacterial and archaeal abundances for key

nitrogen metabolism enzymes

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Nitrite oxidation

~75% Bacteria

Ammonia oxidation

~85% Archaea

Microbial ‘slime’ may represent novel

chemolithotrophic communities

Ammonia

(NH3)

Nitrite

(N02-)

Nitrate

(N03-)

• No detectable organic carbon in cave water around communities

• Communities thought to be lithotrophic – supported by ammonia and nitrite

oxidation, carried out by archaea and bacteria

• Found genes for 3-hydroxypropionate/4-hydrobutyrate pathway used for

carbon assimilation in Nitrosopumilus maritimus

Ammonia

monooxygenase

(AMO)

Nitrite oxidase

Hydroxyl-

amine

(NH2OH)

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SEM images from Weebubbie slime community

Microscopy results

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A&B (controls): S. metallicus and E. coli stained with Arch915

(green) and Eub338(red) probe.

C&D: Weebubbie slime community sample stained with Arch915

and Eub338 probe.

Fluorescence In Situ Hybridization

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Comparative 16S survey- Weebubbie and Warbla

Weebubbie original (metagenome)

Weebubbie 3

Weebubbie 2

Weebubbie 1

Warbla 4

Warbla 3

Warbla 2

Warbla 1

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More weird Australian microbial communities

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Acknowledgements

Samples and pictures

Cave divers responsible for

sample collection (most

pictures from Liz Rogers)

Cave community analyses

Katy Breakwell, Jan

Rieckmann, Liam Elbourne,

Andrew Holmes, Michael

Gillings, Ian Paulsen