Microbial diversity in deep sub-seafloor sediments ... - CiteSeerX

Microbial diversity in deep sub-seafloor sediments assessed by denaturing gradient gel electrophoresis (DGGE) Gordon Webster, Carole J. Newberry, John C. Fry and Andrew J. Weightman. Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, CF10 3TL, UK. ([email protected] ([email protected])) INTRODUCTION n Recent investigations on the deep marine environment have

demonstrated the presence of a significant microbial biomass buried deep within the sediment. n It is now believed that these environments play a major role in the global cycling of elements and contain a large reservoir of organic carbon. n However, little is known about the microbial populations present due to

difficulties in isolating such a physiologically diverse set of microbes and because investigations on this environment are is still in their infancy. n The aim of the following work was to develop rapid molecular microbiological methods (e.g. PCR-DGGE) based on 16S rRNA gene technology to study the diversity of microorganisms in the deep biosphere.

4.15 98.3 193 M

M

7168

7112

7132

7190

Nank15 Nank21

Nank1M C12

C11

27 28 Nank22

METHODS

n Nankai Trough - Hole 1173 on Leg 190 (32 o 14.663’N 135 o 1.509’E) at 4790.7

mbsl, depths 4.15, 98.3 and 193.3 mbsf. n Chilean Continental Margin - Gravity Cores GeoB 7168-3 (37 o 24.00’S 74 o 30.83’W) at 4650 mbsl, depths 0.3, 1.2, 2.25, 3.25, 4.25, 5.25 and 6.25 mbsf; GeoB 7112-3 (24 o 02.00’S 70o 49.41’W) at 2507 mbsl, depth 2.2 mbsf; GeoB 7132-5 (29 o 28.00’S 71o 53.49’W) at 3248 mbsl, depth 1.0 mbsf; GeoB 7190-3 (44o 16.99’S 75o 51.93’W) at 3285 mbsl, depth 3.1 mbsf. DNA extraction and PCR amplification of 16S rRNA genes Small cores (2cm diameter) were aseptically removed from the frozen marine sediment whole round cores and DNA was extracted from 5 g using the FastDNA spin kit for soil. DNA was further cleaned and concentrated using a Microcon YM100 filtration device. Amplification of archaeal and bacterial 16S rRNA genes were performed using the following primer combinations in a nested PCR. n Bacterial 16S rRNA primers : 27F - 1492R (1) and 341F-GC - 518R (4). n Archaeal 16S rRNA primers: Ar3f - Ar9r (2) and PARCH340f - PARCH519r (5). n Euryarchaeal 16S rRNA primers: 1A - 1100A (3) and PARCH340f -

PARCH519r (5).

Fig 2B. DGGE profile of bacterial 16S rRNA genes from deep sea sediment samples from the Chilean Continental Margin.

4.15 mbsf; 98.3 mbsf; 193.3 mbsf; M, DGGE marker (Pseudomonas sp., Staphylococcus sp., Bacillus sp., Arthrobacter sp.).

GeoB 7168-3 (2.25 mbsf); GeoB 7112-3 (2.2 mbsf); GeoB 7132-5 (1.0 mbsf); GeoB 7190-3 (3.1 mbsf); M, DGGE marker.

Table 1 BLAST search results on bacterial 16S rRNA gene sequences from excised DGGE bands Sequence

BLASTN result

% Similarity

Phylum

Environment found

NANKAI1 Nankai 1173 4.15 mbsf

Uncultured bacterium clone MB-B2-103 Uncultured bacterium clone AT425_EubA5 Spirochaeta sp. Buddy

99

OP9

Uncultured bacterium clone LSI

97

Sediment, Forearc Basin (methane hydrate) Sediment, Gulf of Mexico (gas hydrate) River sediment, trichloroethene-dechlorinating culture Methanogenic bioreactor


Carnobacterium sp. FTR-1 Carnobacterium sp. LV62:W11

100 100

Firmicutes

Permafrost, Alaska (psychrotrophic) Lake water column, Antarctica


Unidentified bacterium strain JTB138 Uncultured bacterium clone MB-B2-103 Uncultured bacterium LCK-41 Desulfurellapropionica

98

OP9

Sediment, Japan Sea, cold seep

95 94

Proteobacteria

97

NANKAI1M Nankai 1173 98.3 mbsf

Uncultured gamma proteobacterium WKA19 Serratia proteamaculans strain DSM4543 Serratia proteamaculans subsp. quinovora strain CP6f Serratia proteamaculans strain DSM4597 Marinolactobacilluspsychrotolerans strain 021 Unidentified bacterium F27

C11 Chile 7190-3 3.1 mbsf

Uncultured bacterium clone MB-B2-103 Uncultured bacterium clone CS8.21

98

C12 Chile 7132-5 1.0 mbsf

Uncultured bacterium clone MB-B2-103 Uncultured bacterium clone CS8.21

98

NANKAI22 Nankai 1173 4.15 mbsf NANKAI27 Nankai 98.3 mbsf NANKAI28 Nankai 1173 98.3 mbsf

DGGE and sequence analysis DGGE was carried out as described (6) (Fig. 1). PCR products were separated using 8% polyacrylamide gels with a denaturant gradient between 30 and 60% at 200 V for 5 h. Gels were stained in SYBRGold for 25 min and viewed under UV. Excised DGGE bands were re-amplified by PCR and the re-amplified products were sequenced directly with either 518R ( Bacteria Bacteria)) or PARCH519r (Archaea Archaea)) primer. Partial bacterial and archaeal 16S rRNA sequences were subjected to a NCBI BLAST search to identify sequences with highest similarity.

98 97

Spirochaetes

97

Denaturant Concentration

DGGE

n

n

DGGE utilises a linear gradient of chemical denaturants (urea and formamide) in a polacrylamide gel to separate DNA fragments of equal size based on differences in their nucleotide sequence. As the DNA fragments pass through the denaturing gradient they begin to melt and unzip. Migration slows down until eventually the double strand fully unzips and the GC-clamp holds the DNA in its final position. Final position is determined by base composition. A DNA fragment with a high GC nucleotide content denatures at a higher denaturant concentration than a molecule with a low GC content.

% Similarity

Phylum

Environment found

99 98

Crenarchaeota

Sediment, Mariana Trench Sediment, Japan Trench, cold seep

ARCH23 Nankai 1173 4.15 mbsf

Uncultured archaeon clone No.15 Unidentified Crenarchaeote strain JTB153

99 98

Crenarchaeota

Sediment, Mariana Trench Sediment, Japan Trench, cold seep


Uncultured archaeon clone MN13BT4-97 Uncultured archaeon clone MN16BT2-71

97 97

Crenarchaeota

Deep sea carbonate crust (Anaerobic methane oxidation)


Uncultured archaeon 50-UMH 8% pond Uncultured archaeon clone Ta1c9

95 95

Euryarchaeota

Coastal solar saltern Methane consuming marine sediment


Uncultured archaeon clone 33-FL49A00 Uncultured archaeon clone 33-FL50A00 Uncultured archaeon clone MN16BT2-84

90

Crenarchaeota

Mid ocean ridge sub-seafloor

Euryarchaeota

Uncultured archaeon clone Ta1c9

95

Deep sea carbonate crust (Anaerobic methane oxidation) Coastal Salt Marsh

Unidentified archaeon clone pMC2A203 Uncultured archaeon clone ANME 23

96 96

Euryarchaeota

Hydrothermal vent Sediment, Aarhus Bay, Denmark

ARCH7 Chile 7168-3 0.3 mbsf

Uncultured archaeon clone 2C25 Uncultured archaeon clone Ta1c9

94 94

Euryarchaeota

Coastal salt marsh Methane consuming marine sediment


Uncultured archaeon clone ACE1_A Methonogenium frigidum,

97 97

Euryarchaeota Sediment, coastal meromictic marine basin Ace Lake, Antarctica


Uncultured archaeon 63-A4 Uncultured archaeon 63-A21

99 97

Euryarchaeota

Sediment, Benguela upwelling system

ARCH25 Chile7190-3 3.1 mbsf

Uncultured archaeon 63-A22 Unidentified archaeon clone pMC2A203

97 96

Euryarchaeota

Sediment, Benguela upwelling system Hydrothermal vent

98

96

H M 0.3 4.25 5.25

Forest soil

Arch25

Proteobacteria

Soil, sugar beet rhizosphere

Fermicutes

Marine organisms, Japan sea

Arch6 Arch7

H

1

2

3

Arch5

4

5

6

Arch50 Arch22

Arch22 Arch23

56

Arch12

Arch26

microbial diversity.

Type strain

97 OP9

97

Sediment, Forearc Basin (methane hydrate) Sediment, cold seep Sediment, Forearc Basin (methane hydrate) Sediment, cold seep

CONCLUSIONS n All deep sea sediment samples analysed show a high degree of n Both bacterial and archaeal sequences can be retrieved from

Hailaer soda lake OP9

Figure 3A shows the differences in archaeal populations from 3 different sediment samples from the Chilean Continental Margin. In all samples analysed the Euryarchaea population is smaller than the total population of archaeal sequences found. These findings are important in demonstrating the presence of a group of organism that are responsible for a functional role within the deep biosphere such as methanogenesis. A depth profile of the Euryarchaea (possible methanogenic) community was undertaken on samples from core GeoB 7168-3 (Fig. 3B), interestingly only samples from the depths 0.3, 4.25 and 5.25 mbsf were shown to amplify with the primers used suggesting that only Euryarchaea sequences are present at these depths. Euryarchaeal sequences shown in Figure 2B were confirmed by direct sequencing and demonstrated that some sequences were similar to methanogenic Archaea and also to Archaea that are thought to be involved in anaerobic methane oxidation (Table 2).

Arch53

Type strain

94

Figures 2 and 3 show that there is a high degree of microbial 16S rRNA gene sequence diversity within the deep sediments of the Nankai Trough and the Chilean Continental Margin even though these sediments are known to be low in productivity. The bacterial 16S rRNA gene diversity (Fig. 2) in both geographical locations appears to be more diverse than the archaeal community (Fig. 3) as represented by the number of DGGE bands present. However, both sediment types are dominated by a limited number of intensely stained bacterial 16S rDNA bands that show similarity with uncultured bacterial sequences previously found in deep sea sediments (Table 1).

Total archaeal diversity was also investigated in sediment from the Nankai Trough site 1173 at 4.15 mbsf (Fig. 3C). Crude DNA was diluted at different concentrations and amplified separately. The results suggest that different template DNA concentrations result in different archaeal banding patterns, thought to be due to the ‘diluting out’ of the least abundant archaeal species from the DNA template.

Euryarch

7112 7132 7190 7112 7132 7190

Proteobacteria

98 98

Arch H

Arch24

97

90

Fig. 3 Archaeal 16S rRNA gene diversity

Sediment, Forearc Basin (methane hydrate) Lake water, Alpine Lake Cadagno Thermal environment, Kamchatka

Fig 3A. DGGE profile of archaeal and euryarchaeal 16S rRNA genes from deep sea sediment samples from the Chilean Continental Margin. GeoB 7112-3 (2.2 mbsf); GeoB 7132-5 (1.0 mbsf); 7190-3 (3.1 mbsf); H, archaeal control (Halobacterium sp.).

Fig. 1 DGGE methodology n

BLASTN result Uncultured archaeon clone No.15 Unidentified Crenarchaeote strain JTB153


Fig 2A. DGGE profile of bacterial 16S rRNA genes from deep sea sediment samples from Nankai Trough site 1173.


Sequence ARCH22 Nankai 1173 4.15 mbsf


Nank4

Sediment Samples Sediment samples were collected from different geographical locations in the Pacific Ocean by the Ocean Drilling Program and the PUCK SO156 Cruise.

RESULTS

Table 2 BLAST search results on archaeal 16S rRNA gene sequences from excised DGGE bands

Fig. 2 Bacterial 16S rRNA gene diversity

Fig 3B. DGGE depth profile of euryarchaeal 16S rRNA genes from deep sea sediment samples from the Chilean Continental Margin Core GeoB 7168-3.

Fig 3C. DGGE gel showing differences in observed archaeal community due to DNA template dilution. DNA template, Nankai Trough site 1173.

0.3 mbsf; 4.25 mbsf; 5.25mbsf; H, archaeal control (Halobacterium sp.); M, euryarchaeal control (Methanoplanus petrolearius).

1, 1/50 dilution; 2, 1/100 dilution; 3, 1/200; 4, 1/500; 5, 1/1000 dilution; 6, 1/2000; H, archaeal control (Halobacterium sp.).

deep sediments. n DGGE can be used readily to analyse microbial populations through sediment depth profiles and demonstrate differences in microbial communities at particular depths. n DGGE can also be used to identify groups of organisms that play

a major functional role in the global cycling of carbon in these sediments. n The above work demonstrates that the methods PCR-DGGE can

be used to rapidly screen large numbers of deep sediment samples (ODP leg 201). REFERENCES ACKNOWLEDGEMENTS The authors would like to thank Barry Cragg and John Parkes, University of Bristol for the ODP leg 190 samples and Jens Kallmeyer and Bo Barker Jørgenson, MPI, Bremen and Laurent Toffin and Daniel Prieur, Institut Universitaire Européen de la Mer, Plouzané for supplying samples from the PUCK SO156 Cruise. GW is funded by the NERC M&FMB thematic programme and CJN by the EU DeepBUG project.

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