Diversity of Bacteria and Archaea from the deep sub-surface marine

environment, Nankai Trough (Ocean Drilling Program, Leg 190), through

molecular genetic analysis of structural and functional genes
Carole J Newberry, Gordon Webster, John C Fry and Andrew J Weightman
Cardiff School of Biosciences , Cardiff University, Main Building, Cathays Park
Cardiff, CF10 3TL, Wales, U.K. Tel: +44 29 20876002. E-mail: newberry@cardiff.ac.uk
INTRODUCTION
Deep sub-surface marine sediments have been shown to
contain significant bacterial populations to depths of around
800 metres below sea floor (mbsf). Molecular genetic analysis
based primarily on amplification of extracted nucleic acids
allows an alternative approach to culture based methods,
providing insights into composition, richness and structure of
microbial communities. As part of the Deep Bacteria
Under Ground (DeepBUG) consortium*, aimed at improving
detection and understanding of bacterial processes in deeply
buried marine sediment, sequence analysis of 16S rDNA
structural clone libraries is being used to investigate the types
and diversity of Bacteria and Archaea within a sub-surface
sediment core. Microbial communities within such anoxic
habitats are an important consideration within the global
carbon cycle.

BOX A - METHANOGEN FUNCTIONAL GENE ANALYSIS

METHANOGEN RESULTS

Fig.1. ODP Leg 190: Site 1173 . A methanogen functional clone library was constructed using primers ME1 and ME2, amplifying 0.76 kb
of the -subunit from the mcr gene [1]. The methanogen phylogenetic tree was constructed to include sequences from Leg 190 plus a
selection of methanogen database (www.ncbi.nim.nih.gov).

Site 1173 showed limited methanogen

diversity with three clusters identified
Majority of clones (61%) were within
Nankai cluster 1

100

Whole round core sediment samples were obtained from the

Nankai Trough, off the coast of Japan.

Nankai 1, 4.3 mbsf

No sequences were unique to one

particular depth

Methanobacteriaceae

group includes
Methanobrevibacter
arboriphilus
AF414035

Significantly greater numbers of

Nankai 1 clones found at 4.3 mbsf
and 193.3 mbsf compared with
clones from Nankai 2 and 3, a
greater combined percentage of
which were present at 93.8 mbsf
(see Table 1 below.).

75

100
99
60
100

We are focusing on prokaryotes involved in important biogeochemical processes, such as methanogenesis, acetogenesis
and sulphate reduction. Analysis may also reveal molecular
signatures of novel organisms. Together with 16S rDNA
analysis for Bacteria and Archaea, exploration of functional
gene libraries is allowing more detailed descriptions of
prokaryotic community structure in the deep biosphere.
Methanogens are distinct in the production of co-enzymes
essential for methane biosynthesis. A specific biomarker for
methanogens is methyl co-enzyme-M reductase, the key
enzyme in methanogenesis, catalysing the reduction of methyl
co-enzyme-M, leading to the release of methane. Here
methanogen-specific functional PCR primers were used to
retrieve methyl co-enzyme-M reductase alpha-subunit (mcrA)
genes.

Remaining clones equally divided

(12% each) between clusters 2 and 3

10%

96
54

Methanobrevibacter ruminantium AF41404

Methanothermus fervidus X70765
Methanobacterium bryanti AF313806
Methanobacterium formicicum AF414050
Methanosphaera stadtmanae Af414047
Methanococcusigneus AF414039
Methanococcus jannaschii U67529
Methanococcusthermolithotrophicus Af414048
100
Methanococcus vannielii M16893
Methanococcusvoltae X07793
ODP8-ME2 AF121100
ODP8-ME6 AF121101

Methanococcaceae

Table 1.

91

92

70

62

Nankai 2, 93 mbsf

ODP Leg 190 (Nankai Trough): Site 1173 Methyl Co-enzyme-M

clone library

98

Depth metres below sea floor (mbsf)

Methanosarcinaceae

Methanosarcina barkeri Y00158

Methanosarcina mazeii U22258

100

Nankai 3, 198 mbsf

100

Methanosaetaceae
Methanomicrobeaceae
Methanospirillium

Methanosaeta conciliiAF313802
Methanocorpusculum aggregans AF414034
Methanocorpusculum parvum AF414045
Methanocorpusculum bavaricum AF414049
100
Methanomicrobium mobile AF414044
86
Methanofollisl liminatansAF414041
100
Methanospirillumhungatei AF313805
90 55 Methanoculleus
bourgensis AF414036
Methanoculleus thermophilicus AF313804
Methanopyrus kandleri U57340
99
100

98.3
n = 21

193.3
n= 19

Nankai 1

73%

38%

74%

37

Nankai 2

9%

33%

16%

11

Nankai 3

18%

29%

10%

12

GENERALMETHOD
METHOD
GENERAL

CONCLUSIONS

Forall
allphylogenetic
phylogenetictrees,
trees,sequences
sequenceswere
werealigned
alignedby
byCLUSTAL
CLUSTALW.
W. Trees
Treesconstructed
constructedby
bythe
the
For
neighbour-joiningfunction
functionof
ofTREECON.
TREECON. Bootstrap
Bootstrapvalues
values(>50)
(>50)from
from100
100replicate
replicatetrees
treesare
areshown
shownat
at
neighbour-joining
thenodes.
nodes. Scale
Scalebar
barshows
showssequence
sequencedivergence
divergence(BOX
(BOXAA- -Fig.
Fig.1,1,BOX
BOXBBFig.
Fig.22and
andBOX
BOXCC- -Fig.
Fig.3).
3).
the

Methanogen sequences were

detected using primers specific for
the methyl co-enzyme-M reductase
(mcrA) gene

BOX B - ARCHAEA 16S rDNA ANALYSIS

Fig 2. An Archaea 16S rDNA phylogenetic tree was constructed based on partial sequences obtained from Nankai
Trough 4.3 mbsf , using 21F 958R primer pair [3] .

Majority of sequences were most

closely related to Methanobrevibacter
arboriphilus, a H2 /CO2 utilizing
Archaea

BOX C - Bacteria 16S rDNA ANALYSIS

Bacteria 16S rDNA RESULTS
Table 2. Affiliation of clones from 16S rDNA library

ODPN 73-1-ARCH-8
ODPN 73-1-ARCH-102
Nankai
0.1
cluster 1
CRA8-27cm AF119128
sequences
ODPN 73-1-ARCH-3
fall within
99 ODPN 73-1-ARCH-105
Marine
benthic
100
ODPN 73-1-ARCH-71
group B
APA3-11cm AF119137
57
Cenarchaeum symbiosum U51469
ODPB-A AF121092
100
Nankai
cluster 2
33-F120A00 AF355937
sequences
CRA8-11cmAF119127
fall
73
ODPN
73-1-ARCH-7
within
77
Marine
57
ACA16-9cm AF119144
group 1
ODPN 73-1-ARCH-103
Marine benthic group A
APA2-17cm AF119135
Sulfolobus solfarticus X03235
63
57
Thermoproteus tenax M36474
93
Desulfurococcus mobilis M35966
Thermofilium pendens X14835
100
Nankai cluster 3 - 1 clone
ODPN 73-1-ARCH-72
99 ODPN 73-1-ARCH-126
ODPN
73-1-ARCH-84
99
ODPN 73-1-ARCH-61
Nankai cluster 4 sequences fall
73
within Marine benthic group C
CRA9-27cm AF119129
73
ODPN 73-1-ARCH-106
96 ODPN 73-1-ARCH-106A
100 ODPN 73-1-ARCH-149
Nankai cluster 5
Euryarchaeota
ODPN 73-1-ARCH-11
Pyrococcus abyssi Z70246

Most closely related group of organisms (RDP database rdp.cme.msu.edu)

% represented in
clone library

Gram positive Low G + C Firmicutes, Thermoanaerobacter and relatives, and

Nitrospina sub-division

53.0%

Proteobacteria

14.0%

-Proteobacteria

11.0%

Cyanobacteria and Chloroplasts

8.0%

Planctomyctes and relatives

4.0%

5% were chimeric and excluded from tree analysis, 5% currently unsequenced

Fig. 3. A Bacteria 16S rDNA phylogenetic tree was created based on partial sequences obtained from
Nankai Trough sediment. The clone library was constructed using a nested PCR reaction, initial

PCR primers 27F 1492R[3], nested reaction using 63F 1387R[8]. Restriction analysis of the
clone library (n = 40) gave 11 RFLP patterns (HaeIII and HinfI), representatives of which
were sequenced and used for creation of a phylogenetic tree.
JTB138 AB015269

86

NANK_14

NANK-89
GCA018 AF154105
AT425_EubA5 AY053496
SB-15 AF029043
67

* sequences that have recently been placed

33MB-B2-103AY093469

in a bacterial group associated with OP9

candidate division [10]

NANK_7

10%

CS5.10 AB069793
NANK_17
61

NANK_92

62

NANK_9

100

CS_B013 AF419682
77

NANK_52

65

Nitrospinagracilis Nb-211 L35504

98

Desulfobacterium aniliniDAN237601
Clostridium stecorariumL09176
Moorellathermoacetica M59121
Thermoterrabacterium ferrireducensU76364
87

ARCHAEA RESULTS

- Proteobacteria, a n d
Nitrospina subdivision
Low G + C Gram Positive
Firmicutes

NANK_43

85

Skelotonema pseudocostatum X82155

Cyanobacteria and Chloroplasts

Prochlorococcus marinus AF311217

100

Five clusters identified

63
100

100

NANK_57
Ralstonia pickettii L37367

99

Acidovoraxavenae ssp. avenaeAF137505

100

Most fall into Marine group 1 and Marine Benthic Groups A or B [4]

100
93

NANK-140

- Proteobacteria

Acidovoraxtemperans AF078766

Archaea 16S rDNA analysis recovered

diverse sequences from non-extreme
Crenarchaeota
Euryarchaeota sequences retrieved
were all within the same cluster, most
closely associated with uncultured
archaeon from marine sediment
Archaea 21F, 958R primer specificity
checked with bioinformatics software
PRIMROSE [9]. Methanogen species
recovered from Nankai sediments not
hit with 21F, 958R primer pair.
[http://www. cardiff.ac.u k/biosi/
research/ biosoft]
16S Bacteria rDNA analysis showed
moderate diversity with domination
by sequences most closely affiliated
with either low G+C bacteria
Firmicutes) or Nitrospina sub-division
on comparison with RDP database

96

Chlamydia psittaci AB001798

83
100

100

NANK_22
BD2-16 AB015544

Rhodobacterstrain TCRI 14 AB017799

Spirochaeta halophilus M88722
Pyrococcus abyssi Z70246

Bacteria 16S rDNA phylogenetic tree

shows these sequences clustering with
sequences from a bacterial group
associated with OP9 candidate
division [10]
Other sequences grouped within the
and - Proteobacteria ,
Planctomycetes or were related to
chloroplast genes
Sequences recovered are comparable
with those retrieved from
hydrothermal sediments [10], gas
hydrates [11, 12], cold-seep sediments
[13] and marine sediments from other
locations [14]
ACKNOWLEDGEMENTS

Piruella staleyi DSM 6068T

Remaining clones

*The DeepBUG consortium is a European Union funded project comprising 5 partner institutions covering microbiology,
geochemistry and geology of deeply buried sediments. The research groups involved are: Max Planck Institute for Marine
Microbiology (Marine Biogeochemistry - Prof. B. B Jorgenson), Bristol University (Dept. Earth Sciences/Organic and Biological
Chemistry - Prof. J. Parkesand Prof . R. Evershed), GeoForschungsZentrum (Institute of Petroleum and Organic Geochemistry Prof. Brian Horsfield), Universite de Bretange Occidentale (Microbiology - Prof . Daniel Prieur ), Cardiff University (Environmental
Biochemistry/ Biodiversity and Ecological Processes - Dr A. Weightman/Prof . J. Fry)

-Proteobacteria

NANK_103

96

Fall within Group 1 of the non-extreme Archaea [3]

1. Uncultured marine benthic Crenarchaeota BBA6 - Buzzards Bay sediment [6]

2. Uncultured Crenarchaeota MERTZ_21CM_232 - Antarctic coastal sediments [7]

NANK_137
Acinetobacterlwoffii AY123674

100
83

Majority of sequences recovered were Crenarchaeota

ODP73-1 ARCH-72 was most closely affiliated with two sequences (95%
similarity)

KlebsiellaornithinolyticusY17666
NANK_67

100

96% coverage in clone library - 59 clones

ODP73-1 ARCH-11 and ARCH 149 were most closely affiliated (96% similarity)
with uncultured archaeon TA1e6 from marine sediment, an uncultured
Euryarchaeota [5]

Remainder of sequences were most

closely related to Archaea that use
acetate as a substrate

MA-A2-104 AY093459

95

Number of clones
assigned to each
group

4.3
n = 20

Cluster

Planctomycetales and
relatives

This work was supported by EU project DeepBUG .

Thanks to the Ocean Drilling Program for access
to deep sub-surface sediment cores. Thanks also
to Barry Cragg, John Parkes and Julian Marchesi
for sub-sampling of whole round cores

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