Chemosynthetic symbioses model
based on Solemya velum endosymbiont
Mini-project report
Microbial Diversity Course
Author: Piotr
Starnawski
Preface
Microbial Diversity mini project title:
Chemosynthetic symbioses model based on Solemya velum endosymbiont
Project period:
01.07.2013-25.07.2013
Author:
Piotr Starnawski
Supervisor:
Colleen Cavanaugh, Ph.D.
This report summarizes a mini-project, which was performed in the Marine Biological Laboratory, Woods Hole, during a summer course on Microbial Diversity. The final
report represents a description of the project carried out during the second part of the
course. It consists of 7 chapters, starting with a theoretical background introducing the
reader to the topic of science of this study. The introduction is followed with description
of materials, methods and obtained results with a discussion. Finally a summarization of
work is presented in conclusions followed by future perspectives. The report is finished
with a bibliography and enclosures. References are made according to Harvard referring
system, and are presented in the references section at the end of this report. In the text
all references are marked as follows: (authors surname, year of publication). I would like
to thank the course directors Steve and Dan, all of the Teacher Assistants and course
members for their help and support.
Piotr Starnawski
Contents
1 Theoretical background
1.1 Chemosynthetic Symbioses . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2 Solemya velum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3 Endosymbiont characterization . . . . . . . . . . . . . . . . . . . . . . . .
1
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2
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2 Materials and methods
2.1 Specimens collection . . . . . . . . . . . . . . . .
2.2 Symbiont cultures . . . . . . . . . . . . . . . . . .
2.3 Scanning Electron Microscopy of the gill tissue . .
2.4 Fluorescent In-Situ Hybridization of gill tissue . .
2.5 BLAST search of local databases for the symbiont
2.6 454 gill microbiome . . . . . . . . . . . . . . . . .
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4 Discussion
4.1 Symbiont cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2 Symbiont imaging in the gill tissue . . . . . . . . . . . . . . . . . . . . . .
4.3 454 gill microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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5 Conclusions
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3 Results
3.1 Specimens collection . . . . . . . . . . . . . . . .
3.2 Symbiont cultures . . . . . . . . . . . . . . . . . .
3.3 Scanning Electron Microscopy of the gill tissue . .
3.4 Fluorescent In-Situ Hybridization of gill tissue . .
3.5 BLAST search of local databases for the symbiont
3.6 454 gill microbiome . . . . . . . . . . . . . . . . .
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6 Perspective
25
Bibliography
27
7 Enclosures
7.1 Primers and probes . . . . . . . . . .
7.2 Wizard PCR Preps DNA Purification
7.3 Mo-Bio fast soil DNA extraction kit .
7.4 Media compositions . . . . . . . . . .
7.4.1 Sea Water Base . . . . . . . .
7.4.2 Thiosulfate medium . . . . . .
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System
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Abstract
The aim of this project was first of all to isolate and attempt to cultivate the intracellular
sulfide-oxidizing symbiont of Solemya velum. This was done by dissecting the gills and
cultivating the extract in solid and liquid media in dilution series. Cultures were monitored
with symbiont-specific PCR for the growth of desired organism and all obtained isolates
had their 16S gene sequences. Further work aimed at characterizing the overall bacterial
community found in the bivalves gills using 454 pyrosequencing. DNA form gills from 6
specimens was prepared and additional controls of 2 other clams living in the area were
sequenced and the resulting community was analyzed. In order to better characterize
the symbiosis, Fluorescent In-Situ Hybridization and Scanning Electron microscopy and
techniques were applied for visualization of the symbiont and possible other bacteria living
on the gill tissue. All this information was supplemented by analysis of existing data on
this symbiosis model which comprised of symbiont partial genome and 454 datasets from
sediment known to be occupied by Solemya velum.
1
Theoretical background
1.1
Chemosynthetic Symbioses
The word ”symbiosis” originates from greek and means ”living together” and is used to
describe two di↵erent organisms which live together, regardless if the outcomes are positive
or negative. Chemosynthesis refers to the process of energy conservation using chemicals
(Madigan et. al. 2012). This report focuses on symbioses between bacteria and marine
invertebrate, where the host provides the conditions for the bacterium to grow and provide
fixed carbon which is then used directly or indirectly by the host (Cavanaugh et. al.
2006). The two main types of chemotrophic symbionts observed in marine environments
are methanotrophy and chemoautotrophy - see Table 1.1.
Table 1.1: Marine animals with chemolithotrophic or methanotrophic endosymbiotic bacteria (Madigan et.al., 2012)
Host (genus or class)
Porifera (Demospongiae)
Platyhelminthes (Catenulida)
Nematoda (Monhysterida)
Mollusca (Solemya, Lucina)
Mollusca (Calyptogena)
Mollusca (Bathymodiolus)
Mollusca (Alviniconcha)
Annelida (Riftia)
Common name
Sponge
Flatworm
Mouthless nematode Clam
Clam
Clam
Mussel
Snail
Tube worm
Habitat
Seeps
Shallow water
Shallow water
Vents, seeps, shallow water
Vents, seeps, whale falls
Vents, seeps, whale falls
Vents
Vents, seeps, whale falls
Symbiont type
Methanotrophs
Sulfur chemolithotrophs
Sulfur chemolithotrophs
Sulfur chemolithotrophs
Sulfur chemolithotrophs
Methanotrophs
Sulfur chemolithotrophs
Sulfur chemolithotrophs
Chemosynthesis refers to (i) the ability of an organism to oxidize reduced inorganic
compounds for energy and use it to fix CO2 for biomass, like in case of chemolitotrophs
using sulfide as the receded compound or (ii) the ability to use singe carbon compounds as
both energy and carbon source, like in case of the methanotrophs (Stewart et. al. 2005).
In this study, a focus is placed on the highly sulfidic environment, where the chemolitotrophic endosymbionts are mostly encountered, utilizing the sulfide-rich environment for
their energy metabolism.
Theoretical background
1.2
Solemya velum
Solemyid clams are found throughout the worlds oceans, in shallow water and deepsea habitats, and in temperate and tropical regions. The family diverged between 435
and 500 million years ago, and its members maintain a primitive form even today. The
clam digs very characteristic Y-shaped burrow (Figure 1.1 a), to place itself lower in the
sediment in the sulfide-rich region, while still having access to fresh, oxygenated water.
The relatively recent discovery of symbionts provided the information about the reason
for such behavior. The bacterial endosymbionts supply the animal nutrition derived from
the chemoautotrophic fixation of CO2 fueled by the oxidation of reduced inorganic sulfur
compounds from the Solemya habitat. Indeed, as other Solemya species are reexamined,
they have all been found to harbor bacteria in their gill cells (see Figure 1.1 b).
(a) Y-shaped burrows dug by Solemya velum (From
Stanley 1970)
(b) Transverse section of gill filaments
of Solemya borealis, showing intracellular rod-shaped bacteria (arrows, rectangle) b: bacteriocyte nucleus; c: ciliated
cell nucleus; i: intercalary cell nucleus;
bl: blood space; ci: cilia; mv: microvilli
Figure 1.1: Solemyid clams characteristic features (Cavanaugh et. al. 2006
1.3
Endosymbiont characterization
Symbionts are believed to be integratedgrated into the entire life cycle of Solemya velum
clam, given their presence in reproductive tissue, larvae, juveniles, and adults (Cavanaugh,
1983, Krueger et. al. 1996). It is hypothesized that female hosts transmit the bacterial
symbionts to new generations, and that bacteria derived from this seed population colonize
the gills as filaments are formed in juvenile clams. The functional role of the symbionts
in young Solemya velum hosts remains unknown, however. The symbionts are probably
not metabolically active before and just after spawning, but they colonize the gill buds
and appear to be actively growing while still within the larval egg capsule (Krueger et. al.
1996).
2
2
Materials and methods
2.1
Specimens collection
Specimens for all the studies performed during tho project were collected in the Elizabeths Islands area on the eastern coast of the USA. Sampling was done near Naushon
island (Figure 2.1) in two distinct sampling sites (Figure 2.2). Sediment was collected and
sieved for Solemya velum. Simultaneously other clams were collected to serve as a control
and sediment was collected for keeping the animals in the lab. Organisms were placed in
a plastic container with salt water and transferred to lab. Dissections were done immediately after collection (1-2h) and remaining clams were placed in sediment containers kept
in continuous fresh sea water flow aquarium.
Dissections of organisms were performed using sterile forceps and scissors for each
separate organism. After separating the gill from the rest of the body it was washed in
autoclaved 80 % Sea Water Base (SWB, see Enclosures), weighted and placed in an Eppendorf tube on dry ice for snap-freezing. Frozen gills were stored in -20 °C and used later for
DNA extractions. For the cultivation purposes the gills were not frozen but homogenized
immediately.
2.2
Symbiont cultures
In order to try to obtain an isolate of the gill symbiont, numerous liquid and plate cultures were prepared using the thiosulfate medium with or without yeast extract addition
(0.02 g/l) and gradient tubes with sodium sulfide plug (see Enclosures for media descriptions). The dissected gills were washed in 80 % SWB and then placed in an Eppendorf
tube with 100 µl of the 80 % SWB. Homogenization was done by hand using a plastic
tissue homogenizer for around 2 minutes. The volume of homogenate was then adjusted
to 1 ml with the same solution and used for incubations according to Figure 2.3. A total
number of 5 di↵erent gills were used for starting these cultures. All tubes and plates were
held in dark (cardboard box for the plates and office drawer for gradient and regular tubes)
and monitored daily for growth.
Materials and methods
Figure 2.1: Location of the sampling sites in the Buzzards Bay
Figure 2.2: Precise situation of the two sampling sites for the specimens used in this study
4
Materials and methods
Figure 2.3: Schematic of the inoculations of liquid, solid and gradient cultures with homogenized gill tissue
The monitoring of cultures was done by the use of symbiont-specific primers provided
by Colleen Cavanaugh’s lab in Harvard (see Enclosures for sequences). The monitoring
was performed by PCR method where the colony or 1 µl of liquid culture was added to
the PCR reaction tube. For the reaction mix see Table 2.1.
Table 2.1: PCR mix used to check for symbiont-specific 16S product from the
colonies/liquid cultures
Volume [µl]
6.25
1.00
1.00
3.25
11.50
Compound
Promega Go-Taq Green 2X Mix
Forward primer 120F (15 pmol)
Reverse primer 1612R (15 pmol)
Nuclease-free water
Total volume per sample
The product was then ran on a 1 % agarose gel to check for a product of around 1.5
kbp. For a positive control, purified DNA from gill extracts from Solemya velum used
for 454 preparations were used. As a negative-control samples with MQ water were used.
Selected isolate colonies obtained by re-streaking on solid medium were prepared for 16S
5
Materials and methods
Sanger sequencing to establish the identity of the colony. This was done using the universal
8F/1492R primer pair (see Enclosures for sequence) according to the mix presented in Table
2.2
Table 2.2: PCR mix used to check for bacterial 16S product from gill extracts
Volume [µl]
12.5
2.0
2.0
6.5
23.0
Compound
Promega Go-Taq Green 2X Mix
Forward primer Univ8F (15 pmol)
Reverse primer Univ1492R (15 pmol)
Nuclease-free water
Total volume per sample
Similarly the product was checked on 1 % agarose gel. Purification of the product was
done using the Promega Wizard PCR Preps DNA Purification System according to protocol
(see Enclosures). Purified colony 16S products were then quantified on a NanoDrop system
and send for Sanger sequencing.
2.3
Scanning Electron Microscopy of the gill tissue
Further visualization of the symbiont organism in the gill tissue was done with Scanning Electron Microscopy (SEM). Four organisms were selected and their mantles were cut
under the dissecting microscope. After they were opened, two were placed in 80 % SWB
4 % formaldehyde fixative and two in 1X Phosphate Bu↵ered Saline (PBS), 4 % formaldehyde, 0.5 % glutaraldehyde fixative solution (referred to later as SWB and PBS fixations).
Fixation was carried out for 2 h and after that organisms were washed thoroughly with
their respective bu↵ers (80 % SWB and 1X PBS). In the bu↵er the dissections were done,
where the gill was separated from the body and cut in half with a razor blade. Half pieces
of the gill were placed (under submersion) in plastic containers and remained there for the
rest of the washing steps. The washings were as follows:
1) 3 times 10 minutes in the respective bu↵er (80 % SWB or 1X PBS)
2)
3)
4)
5)
2
3
3
3
times
times
times
times
5 minutes in 25 % EtOH
10 minutes in 50 % EtOH
10 minutes in 75 % EtOH
10 minutes in 100 % EtOH
After that samples were kept in fresh 100 % EtOH and transferred to the critical point
drying machine - Samdri-780A. After drying, samples were placed on metal stages with
carbon surface and coated with 10 nm layer of platinum in vacuum. Afterwards they were
stored overnight in a desicator at room temperature and placed in the SEM for imaging.
6
Materials and methods
2.4
Fluorescent In-Situ Hybridization of gill tissue
In order to visualize the symbiont and it’s location in the gill tissue, Fluorescent In-Situ
Hybridization (FISH) was applied. A specimen of Solemya velum was dissected under a
dissecting scope and the gill tissue was separated from the rest of the body. Then it was
placed in 4 % formaldehyde solution in 80 % SWB for 1 h 30 min. After that a thorough
was with 80 % SWB was performed to remove all formaldehyde and such fixed gill was
stored at 4 °C. The sample for FISH was prepared in a 10-well glass slide. Gill was dissected
further until single ”pages” of the ”book gill” tissue could be seen. These were collected
by forceps and placed in a water drop inside of the slide well. A total number of 10 tissue
pieces were isolated in such way. Slide was then dried at 46 °C to firmly place the gill
tissue inside the well. After that a dehydration step was performed using a series of 3
ethanol baths for the slide: 50 % EtOH, 80 % EtOH and 96 % EtOH, in all cases 2 minute
wash each. After slide has dried o↵ the hybridization was performed by dropping 15 µl of
bu↵er-probe solution to each well. The solution was prepared by mixing the hybridization
bu↵er (see Table 2.4) and the desired probe (EubI-III and Gam42a + Bac42 a competitor
probe in case of this study) in 300:1 ratio (300 µl of bu↵er and 1 µl of 50 ng/µl probe).
Table 2.3: Hybridization and Washing bu↵ers composition used for the FISH of the gill
tissue
Hybridization bu↵er volume [µl] Washing bu↵er volume [µl] Compound
700
Formaldehyde
500
0.5 M EDTA
360
700
5 M NaCl
40
1000
1 M Tris-HCl
1
25
20 % SDS
900
fill-up to 50 ml
MQ water
The slide with the bu↵er-probe solution was placed in a horizontally-situated 50 ml
Falcon tube with a tissue paper soaked in the hybridization bu↵er situated at the end of
the tube. Hybridization was performed for 4 h at 46 °C. After that slide was removed from
the chamber and the wells were washed using the pre-heated to 48 °C washing bu↵er (see
Table 2.4). Then the whole slide was placed vertically in a 50 ml Falcon tube filled with
the hybridization bu↵er and left at a water bath at 48 °C for 15 min. After the washing
slide was removed from the Falcon tube and washed again with MQ water. After the slide
was dried a DAPI staining was done by placing 15 µl of the 1X DAPI stain in each well
and incubating the slide for 10 min in the dark at room temperature. Finally the slide was
washed with MQ water and then with 80 % EtOH and air-dried. During the microscopy
Citifluor solution was applied to enhance the signal.
7
Materials and methods
2.5
BLAST search of local databases for the symbiont
Due to the long history of research in the area and the fact, that the symbiont can be
found in the free-living state a search of existing datasets was performed to find, if sequences
belonging to the symbiont were previously found in the area surrounding Woods Hole. 454
datasets from 2010, 2011 and 2012 were selected for that, where 2010 was a metagenome of a
Little Sipperwisset Salt Marsh microbial mat and the remaining two were large 16S surveys
of various environments. Using the blast+ package ”makeblastdb” command databases
for all thee sets were made. As a query the 16S sequence of symbiont bacterium was used
(GenBank: M90415.1) and additionally a partial genome, provided by Colleen Cavanaugh’s
lab in Harvard, of the symbiont was also tested (68 contigs genome, unpublished). These
were are checked against the database on the nucleotide level (”blastn” command) and the
results were summarized using the circos imaging software package. For the 16S sequence
a percentage-identity cuto↵ of 97 % was used (in order to look for the same operational
taxonomic units) and for the partial genome 93 % percentage-identity and 1e-100 e-value
cuto↵s were used to target highly similar and significant hits.
2.6
454 gill microbiome
For the preparation of the gill microbiome, six Solemya velum, two Tagelus plebeius
and two Yoldia limatula were chosen (the latter two serving as controls not containing the
symbiont). Frozen gills were thawed on ice and then DNA was extracted using the Mo-Bio
PowerSoil DNA Isolation Kit according to protocol (See Enclosures). The only change to
the standard flow was shortening the homogenization step to 1 min due to using a more
thorough homogenizer. Extracted DNA was then checked by performing a PCR reaction
using regular 8F and 1492R (see Enclosures for sequences) primers to see if a product is
obtained. PCR mix is presented in Table 2.2. For the reaction, 2 µl of gill extract was used.
30 cycles of PCR were performed and 5 µl of the product was run on a 1 % agarose gel
with a 1 kb DNA ladder. After checking the result the DNA extract was used to perform
a new PCR with barcoded primers. For each sample, one distinct barcode was selected.
The mixture was prepared according to volumes presented in Table 2.4.
Table 2.4: PCR mix used to obtain a barcoded 16S product from gill extracts
Volume [µl]
15
2.4
0.6
2.4
4.6
25.0
8
Compound
Phusion 2x HF MasterMix
DMSO, 100 %
Forward primer 515F (6.25 uM)
Reverse primer 907R (25 uM)
Nuclease-free water
Total volume per sample
Materials and methods
To each tube 5 µl of gill extract was added and the PCR was run for 30 cycles. Obtained
product was checked on a 1 % agarose gel with a 1 kbp marker. Barcoded products
were pooled, purified and send for sequencing on a 454 machine. Sequences were then
analyzed using Qiime software according to the standard 454 operating procedure. Samples
were separated from the whole plate according to the barcode using the split libraries.py
command. Sequences of quality scores below 25 were discarded, and analysis was performed
on sequences between 400 and 600 bp long. Next step comprised of starting the workflow for
Operational Taxonomic Units (OTU) picking with pick de novo otus.py command which
also assigned RDP taxonomy to picked OTUs. Finally, the results were summarized using
summarize taxa through plots.py command. To check, if the 454 data contained the gill
symbiont sequences an additional analysis was done, where a blast database was prepared
from the 454 data and the 16S gill symbiont sequence (GenBank: M90415.1) was used as
a query against it. Sequences were filtered for 99 %, 98 % and 97 % identity to the query
to look for the exact same sequence or one belonging to the same OTU.
9
3
Results
3.1
Specimens collection
After the collection of organisms they were stored in 1 l plastic containers filled with
sulfidic sediment collected at the same place where organisms were collected.
These containers were covered with a net to avoid organisms from escaping and kept in an aquarium under constant
seawater flow (See Figure 3.1). Dissections were preformed in
order to separate gills that contain the symbiont bacterium.
Organism size was noted and gills were washed in autoclaved
80% SWB and weighted on a laboratory balance. Some of the
extracts were used for starting cultures and some for DNA extractions for 454 pyrosequencing - the summary of dissections
and which samples were used for DNA extracts is presented
in Table 3.1. Other specimens were used for starting cultures
with them in order to cultivate the symbiont. All extracts Figure 3.1: Container used
were kept at -20°C after flash-freezing in liquid CO2
for storing the organisms,
kept in an aquarium with
constant seawater flow
3.2
Symbiont cultures
During the three-week culturing of solid, liquid and gradient cultures of the gill extract
very little growth has been observed. The most prominent changes in the medium have
been served for the gill extract from organism Sv2 where the medium became very milky
in comparison to the starting, transparent solution (Figure 3.4). This was for the first
dilution of the series, 10 1 . This dilution was used for inoculation of two new tubes (with
and without 0.02 g/l yeast extract - YE), to see if further enrichment can be obtained.
Moreover solid plates were also streaked (also with and without YE). After a week no
significant darkening of the liquid cultures was observed, however the plate cultures yielded
growth. These were re-streaked in order to obtain single colonies. It has been observed,
that if a growth occurred on plates containing YE it would still occur, after re-streaking, on
Results
a plate without YE (and vice-versa). During the last week colonies were also observed to
form on the Sv13 and Sv14 plates with YE using the 10 1 dilution. These changed with time
from transparent ones to light-green in the inside indicating a pH change (bromothymol
blue turns from blue color at basic pH to green around neutral and to yellow in acidic),
which can be seen in Figure 3.2 b and c. Colonies from all plates and liquid cultures were
chosen to be screened with a PCR reaction using symbiont-specific primers. The resulting
gel is presented in Figure 3.3 a.
Table 3.1: Summary of dissections performed on all collected organisms. NA = data not
collected. Starred (⇤) samples were used for 454 pyrosequencing
Sample name
Sampling site A
Sv1
Sv2
Sv3
Sv4
Sv5
Sv6
Sv7 ⇤
Sv8 ⇤
Sv9 ⇤
Sv10 ⇤
Sv11 ⇤
Sv12 ⇤
Sv13
Sv14
Tag1 ⇤
Tag2 ⇤
Sampling site B
Yl1
Yl2 ⇤
Yl3
Yl4
Yl5 ⇤
Organism
Solemya velum
Tagelus plebeius
Yoldia limatula
Size [mm]
Gill wet mass [mg]
Comment
NA
14
13
14
NA
NA
18
18
18
14
14
12
12
13
24
24
40.4
20.3
36.4
18.0
19.4
16.9
105.0
38.2
47.2
38.6
48.3
28.8
21.6
17.4
33.7
9.5
Shared
Shared
Shared
Shared
Shared
Shared
NA
NA
NA
NA
NA
23.6
17.8
16.0
10.6
11.4
dissecting
dissecting
dissecting
dissecting
dissecting
dissecting
Shared dissecting tools
Shared dissecting tools
Not rinsed in SWB
Not rinsed in SWB
Gills from two specimens
Part of gut attached
Only one gill
The PCR test with the symbiont specific primers did not gave any result indicating
that the obtained colony or liquid culture can be the symbiont. Nevertheless single colonies
were picked again and the 16S was amplified using the 16S universal primers (see Figure 3.3
b). Samples 3, 8 and 11 from Figure 3.3 b did not give any product and thus were dropped
from further analysis. The positive results were purified and send for Sanger sequencing
to check what kind of organism has been isolated from the gill extracts.
12
tools
tools
tools
tools
tools
tools
Results
(a) Sv2 culture
with a reference
(b) Sv13 10
colonies
1
plate streak with colored
(c) Sv13 10 1 plate zoom
on the colored colonies
Figure 3.2: Positive results observed during the symbiont cultivation attempt
(a) Symbiont-specific PCR test on plate
colonies and liquid cultures. Numbers
are following samples: 1: Sv7 DNA extract (positive control), 2: Sv13 10 1
+YE plate, 3: Sv14 10 1 +YE plate, 4:
Sv2 10 1 -YE re-streak 2, 5: Sv2 10 1
+YE re-streak 1, 6: Sv2 10 1 -YE restreak 1, 7: Sv2 10 1 +YE liquid transfer 1, 8: Sv2 10 1 -YE liquid transfer
1, 9: Sv2 10 1 -YE liquid single colony,
10: Sv13 10 1 -YE liquid, 11: Sv14 10 1
-YE liquid, 12: negative-controll
(b) Colony PCR using universal 16S primers for
Sanger sequencing. Numbers correspond to following samples: 1&9: Sv13 10 1 +YE green colony,
2&10: Sv13 10 1 +YE white colony, 3&11: Sv14
10 1 +YE green colony, 4&12: Sv14 10 1 +YE
white colony, 5&13: Sv2 10 1 -YE re-streak 1
colony, 6&14: Sv2 10 1 +YE re-streak 1 colony,
7&15: Sv2 10 1 -YE re-streak 2 colony
Figure 3.3: Positive results observed during the symbiont cultivation attempt
13
Results
Sanger sequencing results were aligned with the most similar hits in the online Silva
service and the tree was built using the CLC Main workbench. Resulting phylogenetic tree
is presented in Figure 3.4.
Figure 3.4: UPGMA tree of the isolates with their most similar sequences. Isolates are
named in the following fashion: gill isolate medium variation colony type where: str streaked transparent colony, white - white milky colony and col - green colored colony
The analysis of the phylogenetic tree shows, that all of the isolates obtained from
the plates can be classified in the Gammaproteobacteria phylum. Specifically, two main
genus are observed - Endozoicomonas and Pseudoalteromonas. Green-colored colonies
obtained on YE plates from Sv13 and Sv14 gill extracts are most closely related to the
Endozoicomonas, while being most closely related to each other. The strakes transparent
colonies and white colonies from the same plate as the green-colored ones can be considered
to be closely related to Pseudoalteromonas, although here a variation can be observed.
although still, the duplicate samples cluster together.
14
Results
(a) An intact book gill, EHT = 2.5 kV, Mag =
61 X
(b) Gill structure, EHT = 2.5 kV, Mag = 160
X
(c) A zoom out on a sliced gill fragment, EHT
= 2.5 kV, Mag = 281 X
(d) A zoom in on section presented in c), showing the symbionts in the tissue, EHT = 2.5 kV,
Mag = 3720 X
Figure 3.5: SEM pictures of the gill tissue and the symbionts inside of it
3.3
Scanning Electron Microscopy of the gill tissue
A total number of four gill tissue specimens were investigated using a Scanning Electron
Microscope (SEM). Imaging enabled to investigate the structure of the gill, by localizing
and imaging the ciliated and microvillar part of the gill tissue (see Figure 3.5 a-d). Images
b-d were obtained by slicing the intact gills (image a) by a two-sided razor blade, re-coating
and re-imaging. This way a sliced section of the inside of the gill (Figure 3.5 b) could have
been obtained, where the boundary between the two regions of the gill is easily visible. In
the microvillar part of the gill (image c) the symbionts were found which can be seen on
image d.
15
Results
3.4
Fluorescent In-Situ Hybridization of gill tissue
The hybridization on the gill tissue and further imaging on
a fluorescent microscope provided images to locate the symbiont in the gill tissue. First experiment comprised of simple
DAPI staining of the fixed gill tissue - see Figure 3.6. Here
one can see that the large nucleus is stained, but numerous
smaller positive signals can be observed. These are expected
to be the symbionts living inside of the tissue. To confirm
that to the extend possible, two FISH probes were used - one
targeting all bacteria (to confirm symbiont is a bacterium,
EUBI-III probe) and one targeting Gammaproteobacteria (to
confirm that symbiont belongs to that phylum, Gam42a probe
with a Bet42 competitor). In both cases the presence of the
symbiont in the tissue and its belonging to these groups was
Figure 3.6: DAPI stainconfirmed - see Figure 3.7 a-f. Blue color on the figures shows
ing of the fixed gill tissue,
the eukaryotic nuclei (from the DAPI staining) and the green
1000X magnification
coloring indicates where the FISH probe annealed. Overlaid
in FIgures 3.7 b, d and f are the DIC images under transmitted light to show, that bacteria
signals are all inside of the tissue.
3.5
BLAST search of local databases for the symbiont
The screening of the local datasets from previous courses (2010 454 metagenomic data
from a microbial mat, 2011 and 2012 454 16S amplicon data from various student projects)
is summarized in Figure 3.8. Very highly similar hits (97 % identity) to the symbiont 16S
were found in both of the amplicon data sets. Moreover, the partial genome 16-23S operon
has also been receiving a number of hits. Apart from the 16S, the metagenomic data from
2010 has also yielded numerous hits on the 23S region. The second config of the partial
genome which has received a few hits from the amplicon data set has received them in
a region where no protein is annotated. However, because the data was exclusively 16S
fragments, it can be stated with a high degree of probability, that there is an unannotated
16S open reading frame in that region. Summarizing, one could observe, that organisms
falling to the same OTU as the symbiont can be found free-living in locations like Salt
Pond water column, School St. Marsh and the sediment near the Wild harbor. Other
organisms very highly similar to the symbiont can be found in various sediments (Eeel
Pond, Martha’s Vineyard, Wild harbor) and microbial mats (Great and Little Sipperwisset
marsh) all around the Woods Hole area.
16
Results
(a) EUBI-III probe, DAPI + Fluorescence,
400X
(b) EUBI-III probe, DAPI + Fluorescence +
DIC, 400X
(c) Gam42a + Bet42a competitor probe, DAPI
+ Fluorescence, 400X
(d) Gam42a + Bet42a competitor probe, DAPI
+ Fluorescence + DIC, 400X
(e) Gam42a + Bet42a competitor probe, DAPI
+ Fluorescence, 630X
(f) Gam42a + Bet42a competitor probe, DAPI
+ Fluorescence + DIC, 630X
Figure 3.7: Merged channels pictures of the gill tissue taken on a fluorescent microscope and
processed using ImageJ software. Captions describe the probe, channels and magnification
used. Blue color indicates DAPI staining, Green is the specific FISH probe and grey the
transmitted-light DIC images
17
Results
Figure 3.8: Graphical summarization of the BLAST study. Lines connecting parts of
circle represent hits between the two databases, and their location the location of the hit.
Di↵erent datasets are represented with di↵erent colors
3.6
454 gill microbiome
After preparing the gill extracts, the bacterial DNA presence was confirmed by universal
16S primer pair 8F/1492R. Product was obtained for all extracts (See Figure 3.9 a) and
thus a specific PCR was performed with barcodes used for 454 sequencing. In this case a
515F/907R primer pair was used producing a shorter fragment. This was again checked
on a agarose gel and a product of desired size was observed for all Solemya velum samples
(see Figure 3.9 b). The control samples Tag and Yl produced a band of a slightly higher
size than expected (expected being around 400bp) which may be the eukaryotic sequence
product.
18
Results
(a) Gill extract PCR using 8F/1492R universal
primers
(b) Gill extract PCR using 515F/907R
barcoded 454 primers
Figure 3.9: 1 % agarose gel pictures. Sample names are noted at top of well, ’N’ stands
for a PCR negative control containing water
Purified products were sequenced on a 454 machine and sequences were analyzed using
Qiime software. OTU clustering and further classification using the RDP taxonomy results
are summarized in Figure 3.10.
Figure 3.10: Graphical representation of the 454 data taxonomical classification done by
Qiime using RDP database
All Solemya velum gill samples have been reported to contain over 99.9 % of one
organism, that could have been classified only to the class level - they belong in the
Gammaproteobacteria. Interestingly, a very similar result has been obtained for one of the
Tagelus plebeius clam samples, whereas the other sample has a much more diverse micro
19
Results
biome. The Yoldia limatula samples on the other hand were found to contain sequences
of unknown origin. Because the extend of information provided by Qiime was insufficient
to confirm, that the gills contain only the symbiont, additional blast check was performed,
which results are presented in Table 3.2.
Table 3.2: Summary of the blast result, where 454 sequencing results were used as a
database and 16S of the gill symbiont as a query
Sample name
Sv7
Sv8
Sv9
Sv10
Sv11
Sv12
Tag1
Tag2
Yl2
Yl5
Total reads
Total reads
22,723
23,983
42,164
55,938
51,653
45,971
746
72
2,296
248
244,891
99 % identity [%]
45.69
53.96
48.34
48.69
46.37
46.46
35.66
1.39
0.13
0.40
99 % identity [%]
99.19
99.83
99.58
99.72
98.82
99.92
93.57
4.17
0.17
0.81
99 % identity [%]
99.30
99.85
99.63
99.77
98.95
99.94
93.83
4.17
0.17
0.81
The results clearly show, that the Solemya velum gill is populated specifically by the
previously reported symbiont, and not by any other bacterium. Already at 98 % similarity
nearly all sequenced fragments map on the published symbiont 16S sequence. What is
very interesting, Tagelus plebeius sample 1 appears to also have the majority of sequences
identical to the symbiont found in Solemya velum. The second sample of this clam has a
significantly lower percentage of hits, but one has to consider the overall low amount of
obtained sequences.
20
4
Discussion
4.1
Symbiont cultures
The culturing of the symbiont attempts yielded a few colonies, which were then screened
with the symbiont-specific 16S PCR and this was followed by by sequencing their 16S gene.
Results from this part suggest, that the colonies that grew on the plates are not necessarily the symbiont (because of the lack of the specific product during PCR). However, the
obtained colonies can be classified into two groups:
Transparent, small colonies which were classified to cluster inside Pseudoalteromonas
genus. These organism are very commonly found in seawater, sediment, sea ice, surfaces of
stones, marine algae, marine invertebrates, and salted foods. They are chemotrophs possessing strictly aerobic metabolism with oxygen as a terminal electron acceptor (Bowman
and McMeekin, 1995). This is in line with the growth conditions used in the study, where
the symbiont cultures were kept aerobically and the chemical compounds that would be
necessary for growth could have came from either the gill inoculate (growth was observed
from 10 1 cultures, where there is still organic matter from the grinder gill) or from the
low amount of yeast extract added to some of the media. Inoculum was also washed in
sterile sea-water, but the washing was possibly not rough enough to remove all organisms
that could reside on the gill tissue.
The second group are colored colonies that grew from inoculates from organisms Sv13
and Sv14 on the yeast extract plates. These organisms were found to classify very well
inside of Endozoicomonas genus. These organisms were previously reported to be isolated
from sponges, sea slugs, corals and starfishes. They are characterized as aerobic or facultative anaerobes performing carbohydrate fermentation (Nishijima et. al., 2013). Some
of the species inside of the genus have been reported to be capable to reduce nitrate to
nitrite (Kurahashi and Yokota, 2007). In all studies they were isolated from gastrointestinal tracks, which could suggest, that during the dissection of the gills a part of the gut of
Solemya velum was also removed and grinder with the gill. The other possibility would be
that these organisms are found also free living in the environment, as the 454 sequencing
of the gill tissue did no yield their sequences inside of the gill.
Discussion
4.2
Symbiont imaging in the gill tissue
The imaging of the bacteria inside of the gill tissue was successful to the extend of
confirming the presence of of the organism inside the tissue and nowhere else. Fluorescent
In-Situ Hybridization was able to confirm the belonging of the symbiont to Gammaproteobacteria and the location inside of the tissue, however in order to be absolutely sure
one would have to design a specific probe targeting the symbiont 16S. Scanning Electron
Microscopy proved to be a very useful technique to image the location and orientation of
the cells inside of the gill tissue, however the full structure of the bacteriosomes could not
be easily visualized. Based on previous studies, the cells should be oriented perpendicular
to the plane of the gill (Cavanaugh et. al. 2006) whereas in this study only a few images
of co-oriented cells were obtained. One of the reasons may be the time of fixation, which
could have been too long. Also the method of sectioning the gill with a razor-blade may
have been to invasive, destroying the sliced surface.
4.3
454 gill microbiome
The data obtained from the gill micro biome 454 sequencing confirmed in a very strong
manner the presence and prevalence of the symbiont is the gill tissue. The usage of Qiime
workflow did not provide however conclusive results, as the taxonomic classification of the
sequences was not deep enough. Confirmation of the sequences belonging to the symbiont
OTU done by blast yielded also some interesting results in regard to the control razor clam
Tagelus plebeius, where the symbiont sequences were found in large numbers in one of the
samples. Specimens of this clam were collected at the same place as Solemya velum used in
this study and moreover it is reported to also burrow inside of the sediment (up to around
10cm) and use the siphon holes (Holland et.al., 1977). The fact that they both clams burrow
inside of the sediment and that the sediment is highly sulfidic could indicate the possibility
of the Tagelus plebeius also possessing the symbionts in its gills. Moreover, the partial
genome of the symbiont (courtesy of Colleen Cavanaugh group in Harvard) suggests a freeliving organism (due to large genome size and transposable elements in the genome) and
thus it could be picked-up by similar organisms in the environment. Arguments against this
theory would be the possibility of cross-contamination of the samples during the laboratory
procedures, the low number of 454 reads in comparison to symbiont-positive samples and
the lack of clear product when regular 16S PCR was performed on the gill extracts. One also
has to remember, that because the symbiont is free-living (which has been also confirmed
by the blast-screen of datasets obtained form local 454 surveys) it can also reside on the
gill and be picked-up if the washing was not thorough enough.
22
5
Conclusions
The outcomes from this project have proven to be very interesting and insightful,
giving a better understanding of the symbioses between the host and bacterium. Culturing
attempts resulted in pure cultures, obtained from agar plates, of a typical marine bacterium
and a symbiont, which has been associated with the gut but not with the gill. Liquid
and gradient tubes did not result in any substantial results. The Fluorescent In-Situ
Hybridization and Scanning Electron Microscopy provided a solid proof of the presence
of the symbiont in the gill tissue and of the size and orientation of its colonies. Finally
the 454 sequencing and data mining have proven the presence of organisms highly similar
to the symbiont (same Operational Taxonomic Unit) in sediments and sea waters around
Woods Hole area, proving the concept of symbol being a free-living organism. Moreover
the 454 sequencing of the gill micro biome also confirmed the identity of the symbiont and
that it is the only bacterium found inside of the gill tissue. A very interesting outcome was
the finding of symbiont sequences inside of Tagelus plebeius gill tissue, however this would
have to be confirmed by other techniques.
Overall, the project was a very successful study which opens grounds for new research
to be conducted, and provides data which can be analyzed further and in parallel with
other studies.
6
Perspective
The information obtained from this study opens grounds for numerous follow-up studies.
One of the two main subjects to be investigated would be the colored colonies obtained
from the gill extracts on yeast-extract containing plates. Although they are closely related
to gut associated symbionts, one could try to characterize them further, as they were
obtain from gills. Cultivation on media without yeast extract would be a good control, to
check if they utilize organic matter or are they fixing carbon dioxide. Second suggestion
would be to check their ability to reduce nitrate to nitrite, which was reported in literature
(and because the cultivation media contains nitrate). Finally one could also try to make
a culture from the gut separately and separately from the gill. This could provide more
information on whether they are in fact obtained from gills or not. The second main
subject worth pursuing would be the Tagelus plebeius clam and the possibility of it hosting
the symbiont in the gill. A more broad screening of the clams would be one way to pursue
this goal, as well as applying imaging techniques used in this study to the gill tissue of this
clam. In case of the hybridization imaging techniques, a symbiont specific probe would be
suggested, after applying simple approaches like DNA-staining just to see, if any bacterial
cells can be found in the tissue.
Bibliography
Kurahashi M. and Yokota A., (2006). Endozoicomonas elysicola gen. nov., sp. nov., a gammaproteobacterium isolated from the sea slug Elysia ornata, Systematic and Applied Microbiology 30:202-206
Holland A. F. and Dean J. M. (1977). The Biology of the Stout Razor Clam Tagelus plebeius: I. Animal-Sediment Relationships, Feeding Mechanism, and Community Biology,
Chesapeake Science 18:58-66
Nishijima M., Adachi K., Katsuta A., Shizuri Y. and Yamasato K. (2013). Endozoicomonas
numazuensis sp. nov., a gammaproteobacterium isolated from marine sponges, and
emended description of the genus Endozoicomonas Kurahashi and Yokota 2007 International Journal of Systematic and Evolutionary Microbiology 63:709-714
Bowman J. P. and McMeekin T. A. (1995). Pseudoalteromonas description in Bergeys manual of systematic bacteriology, volume 2
Cavanaugh C. M., McKiness Z. P., Newton I. L. G. and Stewart F. J. (2006). Marine
Chemosynthetic Symbioses, The Prokaryotes 1:475-507
Krueger D. M., Gustafson R. G. and Cavanaugh C. M. (1996). ?Vertical Transmission of
Chemoautotrophic Symbionts in the Bivalve Solemya velum (Bivalvia: Protobranchia)
The Biological Bulletin 190:195-202
Eisen J. A., Smith S. W. and Cavanaugh C. M. (1992). Phylogenetic Relationships of
Chemoautotrophic Bacterial Symbionts of Solemya velum Say (Mollusca: Bivalvia) Determined by 16S rRNA Gene Sequence Analysis Journal of Bacteriology 3416-3421
Madigan M. T., Martinko J. M., Stahl D. A. and Clark D. P. (2012). Brock Biology of
Microorganisms, 13th Edition, Pearson Education, Inc.
7
Enclosures
7.1
Primers and probes
Name
8F
1492R
515F
907R
120F
1216R
EUBI-III
Gam42a
Bet42a
7.2
Sequence
5’-AGAGTTTGATCCTGGCTCAG-3’
5’-GGTTACCTTGTTACGACTT-3’
5’-CGTATCGCCTCCCTCGCGCCATCAGkNNNNNNNNNkGAkGTGYCAGCMGCCGCGGTAA-3’
5’-CTATGCGCCTTGCCAGCCCGCTCAGkGGkCCGYCAATTCMTTTRAGTTT-3’
5’-ACCTAGTAGTGGGGGACAACTACCG-3’
5’-CGAATCTGAACGCGAGACCCGT-3’
5’-GCWGCCWCCCGTAGGWGT-3’
5’-GCCTTCCCACATCGTTT-3’
5’-GCCTTCCCACTTCGTTT-3’
Wizard PCR Preps DNA Purification System
1. Aliquote 100 µl of Direct Purification Bu↵er into a 1.5 ml Eppendorf tube and add
30-300 µl of the PCR product
2. Vortex briefly to mix and add 1 ml of Resin
3. Vortex 3 times over 1 minute period, place the mini column on the tip of syringe
barrel and place the whole assembly into a vacuum manifold
4. Pipet the DNA/Resin mix to the syringe barrel and apply vacuum to draw the
DNA/Resin into the mini column
5. Add 2 ml of 80 % isopropanol to syringe barrel and apply vacuum to wash the
column. Dry the resin by continuing to draw the vacuum for 30 seconds
6. Remove the syringe barrel and transfer the mini column to a new 1.5 tube
7. Centrifuge the mini column at 10,000 g for 2 minutes to remove isopropanol
8. Transfer the mini column to a new 1.5 ml Eppendorf tube and apply 50 µl of
Nuclease-free water or TE bu↵er to the mini column and wait for 1 minute
9. Centrifuge the mini column at 10,000 g for 30 seconds and then remove and discard
the mini column
Enclosures
7.3
Mo-Bio fast soil DNA extraction kit
1. To the PowerBead Tubes provided, 0.25 grams of soil sample
2. Gently vortex to mix
3. Check Solution C1. If Solution C1 is precipitated, heat solution to 60 °C until
dissolved before use
4. Add 60 µl of Solution C1 and invert several times or vortex briefly
5. Secure PowerBead Tubes horizontally using the MO BIO Vortex Adapter tube holder
for the vortex
6. Make sure the PowerBead Tubes rotate freely in your centrifuge without rubbing.
Centrifuge tubes at 10,000 g for 30 seconds at room temperature
7. Transfer the supernatant to a clean 2 ml Collection Tube (provided)
8. Add 250 µl of Solution C2 and vortex for 5 seconds. Incubate at 4 °C for 5 minutes
9. Centrifuge the tubes at room temperature for 1 minute at 10,000 g
10. Avoiding the pellet, transfer up to, but no more than, 600 µl of supernatant to a
clean 2 ml Collection Tube (provided)
11. Add 200 µl of Solution C3 and vortex briefly. Incubate at 4 °C for 5 minutes
12. Centrifuge the tubes at room temperature for 1 minute at 10,000 g
13. Avoiding the pellet, transfer up to, but no more than, 750 µl of supernatant into a
clean 2 ml Collection Tube (provided)
14. Shake to mix Solution C4 before use. Add 1200 µl of Solution C4 to the supernatant
and vortex for 5 seconds
15. Load approximately 675 µl onto a Spin Filter and centrifuge at 10,000 g for 1
minute at room temperature. Discard the flow through and add an additional 675 µl of
supernatant to the Spin Filter and centrifuge at 10,000 g for 1 minute at room temperature.
Load the remaining supernatant onto the Spin Filter and centrifuge at 10,000 g for 1 minute
at room temperature
16. Add 500 µl of Solution C5 and centrifuge at room temperature for 30 seconds at
10,000 g
17. Discard the flow through
18. Centrifuge again at room temperature for 1 minute at 10,000 g
19. Carefully place spin filter in a clean 2 ml Collection Tube (provided). Avoid
splashing any Solution C5 onto the Spin Filter
20. Add 100 µl of Solution C6 to the centre of the white filter membrane. Alternatively,
sterile DNA-Free PCR Grade Water may be used for elution from the silica Spin Filter
membrane at this step
21. Centrifuge at room temperature for 30 seconds at 10,000 g
22. Discard the Spin Filter
31
Enclosures
7.4
7.4.1
Media compositions
Sea Water Base
Component
NaCl
MgCl2 6 H2 O
CaCl2 2 H2 O
KCl
7.4.2
Final Concentration [mM]
342.20
14.80
1.00
6.71
Thiosulfate medium
Component
Before autoclaving
NaSO4
NH4 Cl100X
K2 HPO4 100X
SWB
For solid medium
Bromothymol blue
Agar
After autoclaving
Na2 S2 O3
NaHCO3
MOPS bu↵er pH 7.2
Vitamin solution 1000X
Trace element solution 1000X
Amount
30 mM
1ml/l
0.1 ml/l
up to 1l
0.02 g/l
15 g/l
5 mM
10 mM
10 mM
1ml/l
1ml/l
33