Hannah Holland-Moritz
Microbial Diversity Final Report
August 15, 2016
Investigating the presence of Nitrogen-fixing Chlorobi in Trunk River's “Lemonade”
Microbial Blooms
Background
Nitrogen is critical and often limiting nutrient in biological systems. The only known sources of
biologically fixed nitrogen are microbial. Nitrogen fixation can be particularly important in
estuarine ecosystems especially during eutrophication events when large blooms of
microorganisms may consume normally plentiful nutrients[1]. In the 2015 Wood's Hole
Microbial Diversity course a yellow bloom “Lemonade” phenomenon was observed at the Trunk
River site [geographic coordinates here]. The bloom was replicable by digging a small trench in
the sediment at the bottom of the pond and waiting for 2-3 days. 16S rRNA analysis from that
year revealed that the major contributing clades to bloom and the source of the yellow coloration
were members of the Chlorobi (Green Sulfur Bacteria) phyla (report from last year/poster).
Subsequent shotgun metagenomics revealed the presence of nitrogenase in each of the major
Chlorobi species. In this project, we attempt to use nitrogen-free enrichment culture, acetylene
reduction assays, qPCR of nitrogen fixation genes and HCR-FISH (Hybridization Chain
Reaction Fluorescence In-Situ Hybridization) to investigate the nitrogen-fixation capacity of
Chlorobi in the Trunk River “lemonade” blooms.
Materials and Methods
Sample Collection
Samples were collected over several days. The first samples, I1 and L2, were collected by
inserting a syringe below the water line to the microbial bloom (“lemonade”) and collecting
50ml. Later samples (IP) were collected at four different depths, two planktonic samples (IP1
and 2) and two sediment samples (IP3 and IP4). For a positive control (SWC), Chlorobi were
scraped off microbial mats that were grown in a constant-flow seawater table. Pseudomonas
aeriginosa was used as a negative control in qPCR and FISH analyses of diazotrophy.
Environmental Measurements
For media design, salinity and pH of the sample collection site (Trunk River) were measured
with an environmental pH and Salinity probe.
Enrichment Cultures
Six enrichments for nitrogen fixing Chlorobi (Green Sulfur Bacteria) were prepared and
inoculated with one milliliter of I1 and L2 respectively. The media was designed using a
modified nitrogen-free version of the Green Sulfur Bacteria recipe found in Volume VII of The
Prokaryotes [2] adapted for the salinity and pH of the inoculation site. The media was then
transferred to serum bottles under anaerobic conditions and the head-space above the media was
filled with a 20%/80% CO2/N2 mixture. The media recipe is as follows:
For 3 liters of media:
Stock Solutions (taken from 2016 MBL Microbial Diversity course manual):
1000x EDTA Chelated Nitrogen-fixing Trace Elements Solution (adapted from the 2016
Microbial Diversity course manual)
Add components in order and wait for each to dissolve before adding the next.
This may take several hours or overnight, in some cases.
Component
Amount
FW
1000x Conc.
Final Conc.
Deionized water
987ml
Na
Na
na
EDTA
5200 mg
Na
20 mM
20 µM
18 mM
18 µM
Adjust pH to 5
*This took us
with 5 M NaOH* close to 20ml
FeCl3 · 6H2O
2023 mg
278.01
7.5 mM
7.5 µM
H3BO3
30.0 mg
61.83
0.48 mM
0.48 µM
MnCl2 · 4H2O
100 mg
197.91
0.5 mM
0.5 µM
CoCl2 · 6H2O
190 mg
237.93
6.8 mM
6.8 µM
NiCl2 · 6H2O
24.0 mg
237.69
1.0 mM
1.0 µM
CuCl2 · 2H2O
2.00 mg
170.48
12 µM
12 nM
ZnCl2
68.0 mg
287.56
0.5 mM
0.5 µM
Na2SeO3 · 5H2O
6.00 mg
263.01
23 mM
23 µM
Na2MoO4 · 2H2O 1.20 g
241.95
5 mM
5 µM
NaVO3
122.00
5 mM
5 µM
0.61
Prepare Media:
51 mL of 1x Seawater base (SWB)
30 mL of 100x Freshwater Base (FWB)
30 ml of 100mM K Phosphate pH 7.2
3 ml of N-fixing Trace Elements solution
Autoclave in Widdel vessel
Cool under stream of N2/CO2 (80%/20%) gas
After cooling, add:
3 ml of 1000x 13-Vitamin Solution, filtered
42 ml of 1M NaHCO3, filtered, or autoclaved under carbon dioxide environment
720 mg of NaS, to 3mM final concentration
30 ml of 1M thiosulfate solution
50 mg of DCMU, to suppress cyanobacterial growth
Dispense into serum bottles under N2/CO2 (80%/20%) atmosphere
Cultures were inoculated with 1ml of sample and left in the dark for 2 hours before being
incubated at room temperature under 660 nm wavelength light (“cherry red”).
Acetylene Reduction Assay
For acetylene reduction assays, 37 mL serum bottles were evacuated three times and filled with 3
mL of inoculum. The vacuum in the bottle was filled to 1 atm of pressure with acetylene using a
syringe, pre-washed with nitrogen. Initial measurements were then taken immediately by
removing 250 µL of gas from each bottle and measuring its retention time by Gas
Chromatography in comparison to a standard containing 15 ppm Acetylene and 15 ppm
Ethylene. Subsequent measurements were taken after 4.5 hours of incubation in light at 660 nm
wavelength (“cherry red”).
Probe and Primers
For mono-FISH identification of chlorobi, the GSB532-ATTO488 probe was used [3]. For HCRFISH [4] oligo probes were designed to target general NifH sequences and the specific NifH
sequences derived from the Chlorobi in the Trunk River metagenomes. For the general NifH
probe we used Nh21F, it was chosen both for it's short length and low-discrimination as
described in Gaby and Buckley 2012. The sequence was 5' – GCI WTY TAY GGN AAR GGT
ATA GCA TTC TTT CTT GAG GAG GGC AGC AAA CGG GAA GAG – 3' . The Chlorobispecific probe sequence was designed in using ARB [5] from an alignment of the metagenomederived Chlorobi NifH sequneces 5' – ATG TGC TTG GTG ACG TTG TGT ATA GCA TTC
TTT CTT GAG GAG GGC AGC AAA CGG GAA GAG – 3'. The Chlorobi-specific NifH
sequence was derived from the same alignment as the corresponding HCR probe. The forward
primer sequence was 5' - ATG TGC TTG GTG ACG TTG TG – 3' and the reverse primer
sequence was 5' – TGT TGT TGG CWG CGT ACA TG – 3'. The Chlorobi-specific NifH probe
and primers were named C-nifH-HCR and C-NifH respectively.
Fluorescent In-Situ Hybridization (FISH)
For mono-FISH the protocol from the 2016 Microbial Diversity Manual was used, the protocol
from the manual is as follows [6]:
For HCR rRNA FISH, the protocol was derived from Yamaguchi et al, 2015 [4]. First
hybridization chambers were prepared using a 50ml conical, a folded kim-wipe and a parafilmcovered glass microscope slide. Atmosphere buffer to maintain the formamide concentration of
the hybridization chamber was prepared for each chamber by mixing the relevant formamide
concentration (see Table 2) with deionized, nuclease-free water. For all incubations, an oven was
prepared to 35 ºC. Hybridization solution was prepared by mixing CARD-FISH amplification
buffer (for 20ml: 3.6mL of 5M NaCl; 0.4 mL of 1M Tris HCl, pH 8.0; 20 µL of SDS (20% w/v);
2 mL of blocking solution; 14mL of sterile milliQ water and fomamide mixed according to the
necessary final formamide percentage such that the percentage of formamide in water is two
times as concentrated as the final percentage desired – see Table 2; 2 grams of dextran sulfate;
heat to between 40 and 60 ºC until the dextran sulfate as dissolved completely – this may take
several hours) with the initiator probe solution (probe concentration of 10uM in sterile, nucleasefree water).
To begin the first incubation, 2µL of probe solution were mixed with 18 of CARD-FISH buffer
of the appropriate formamide percentage on the parafilm-covered slide and filters were drawn
through the droplets. The slide was then placed in the hybridization chamber at 35 ºC for 2 hours.
Washing buffer and a second hybridization chamber with a sterile, nuclease-free water
atmosphere were prepared during the incubation time (for 50ml of washing buffer: 1 ml TrisHCl, X ml 5 M NaCl?, 25 µl 20% SDS, fill up to 50 ml with MilliQ water) and pre-warmed to
35 ºC.
Table 2: Probes with corresponding formamide percentages for hybridization buffer and ml of
5M NaCl for washing buffer
Probe
Final Formamide Percentage Amount of 5M NaCl in
Washing Buffer
C-NifH-HCR
15
3080 µL
NifH-HCR
20
2150 µL
EUB35-HCR
35
700 µL
For the second hybridization, Hybridization II buffer was prepared as follows:
2 ml 500 mM Na2HPO4
3.6 ml 5 M NaCl
10 µl 20% SDS
2 ml 10% blocking reagent
2 g dextran sulfate
12.39 ml MilliQ
water
Total volume: 20 ml
Following the first hybridization, the filters were incubated at 35 ºC for 30 minutes.
90 µL of each hairpin stock solution (100 µM) were mixed with 100 µL of Hybridization II
buffer and heated in a thermocycler at 95 ºC for 1 minute and 30 seconds and allowed to cool
down at room temperature for several minutes.
As soon as filters were removed from the washing buffer 5-10 µL of the warmed hybridization II
buffer-hairpin mix was spread on the filters. Filters were then incubated in their hybridization II
chambers at 35 ºC for 2 hours.
Filters were subsequently washed for 10 minutes in 1x PBS, pH 7.4 at room temperature before
being washed in water and 96% ethanol for 30 seconds each and dried.
For dual-hybridization (mono-FISH and HCR-FISH) mono-FISH reactions were performed first
in all cases, since they were the more stringent hybridization conditions, filters were stored
overnight at -20 ºC and HCR-FISH was performed the following day.
After all hybridizations were complete, filters were DAPI stained, before imaging.
qPCR
DNA was extracted using the Power-Soil DNA extraction kit according to the manufacturer's
instructions (MoBio Laboratories, Carlsbad, CA). C-NifH primers were tested and yielded
successful amplification in a gradient PCR on the positive control SWC sample. The reaction
mix was prepared using Promega Go-Taq Master Mix (Promega Corporation, Madison, WI)
according to the manufacturer's instructions with 10 µM primer working solutions. The
thermocycler conditions were 95 ºC for 2 minutes, 25 repeated cycles of 95 ºC for 30 seconds,
annealing gradient between 45-65 ºC for 30 seconds, 72 ºC for 1.5 minutes followed by 72 ºC for
10 minutes. The most brightest PCR products were around 60 ºC.
For qPCR, Promega's GoTaq qPCR Master Mix (Promega Corporation, Madison, WI) was used
according to the manufacturer's instructions. Thermocycler conditions were set according to the
manufacturer's recommendations, with annealing temperature of 60 ºC. PCR product from the
SWC sample was used as a standard for the qPCR reactions.
Results and Discussion
Enrichments
After two days cultures were still clear but gas was being consumed by the organisms. The headspace was changed every two days on three of the L1 cultures but left intact over two others.
Critically, after four days, the bottles in which the head-space was intact began to show green
aggregates, while the bottles that had the atmosphere perturbed displayed little to no growth.
Eventually, we decided to stop changing the head-space in all bottles and green filamentous
bacteria began to appear in all unperturbed bottles after about four days (Figure 1). Mono-FISH
of these bacteria with the chlorobi probe indicated that chlorobi had been successfully grown and
qPCR of the Chlorobi-specific NifH gene was highest in the enrichment sample (Figure 2).
Acetylene Reduction Assay
Acetylene reduction was measured on the L2 culture a day after collection. The head-space of 37
mL serum bottles was completely filled with acetylene and 3 mL of culture. Initial retention
times were taken for acetylene and ethylene and compared using a paired t-test. Although
acetylene concentrations slightly decreased and ethylene concentrations slightly increased after a
4.5 hour incubation period (Figure 3), only the change in ethylene was significant (p = 0.0303,
acetylene p-value = 0.2631). The lack of significant change in acetylene may be explained by the
high concentration of acetylene in the bottles. A significant change may have been more
observable if the incubation had lasted long enough to appreciably remove acetylene from the
head-space.
qPCR
qPCR of Chlorobi-specific NifH gene (C-NifH) was performed on DNA extracted from a depth
profile of the blooms (samples 1P1-1P4) as well as on the sea water table sample as a positive
control (SWC), the culture inoculum (L2) and the enrichment after a week of unchanged headspace (I1A). Figure 2 shows the results. Though the enrichment and sea water table samples
display high concentrations of NifH, the depth profiles show little or none. IP3, is the only depth
profile sample that shows any hint of Chlorobi-specific NifH, thus we conclude that if there are
chlorobi in the Trunk River blooms this year, they may be coming from the upper layer of
sediment.
HCR-FISH of Inoculum
The L2 sample was prepared for HCR-FISH of Eubacterial probe, NifH, and C-NifH. Although
there was no proof of C-NifH hybridization, the general NifH probe did hit some filamentous
bacteria (Figure 4). These may be contamination from cyanobacteria higher in the water column
or some other nitrogen-fixing organism, though it is also possible that the probe is nonspecifically binding something else. Nevertheless, it is an interesting finding and worth trying to
recreate.
Dual Hybridization of Depth Profile
After the partial success of the NifH probe, a second set of samples was prepared from a depth
profile. These samples were dual-hybridized, first with mono-FISH for Chlorobi, and then with
HCR-FISH for NifH and C-NifH. Unfortunately, these samples were unsuccessfully filtered.
Very few cells made it onto the filters and staining was poor in all areas. To really be certain of
the hybridization, this experiment must be performed again.
Conclusions
Taken together results call into question the presence of a high concentration of Chlorobi in the
lemonade blooms in Trunk River. Although the differences from year-to-year may be due to
different sampling techniques, it is impossible to rule out the possibility of yearly variation given
the data in this report. That, Chlorobi are present, however is obvious given the success of the
enrichments and the qPCR results give good evidence that the Chlorobi that were enriched for
possess the nitrogen fixing gene and may be nitrogen-fixers themselves, since their media
contained only gaseous nitrogen. Acetylene reduction assays also indicate that it is likely that
nitrogen fixation is happening in the inoculum, though which organisms are doing it is
impossible to determine from these data. It is also impossible to say for certain whether the
enriched organisms are major players in the lemonade blooms or whether they actively fix
nitrogen in the environment.
Additionally, qPCR results indicate that the most likely harbor for the nitrogen-fixing Chlorobi in
the depth profile is the first layer of sediment. It would be interesting to follow up on this and
study the source of these organisms as blooms develop and progress.
FISH results indicate that it may be possible to use NifH as probe with the HCR technique but
much more research and verification must go into this process before any final conclusions can
be drawn.
Acknowledgments
The author would like to thank and acknowledge the hard work and contributions of Microbial
Diversity teaching assistants especially Katherine Hargreaves who assisted with the development
and testing of the C-NifH primers, Elise Cowley who helped with sample collection, Kyle Costa
who assisted with the qPCR analysis, Grayson Chadwick and Bonita Lam who helped set up and
run the enrichments, and Dimitri Meier who designed the primers and assisted in all the FISH
techniques. Additional thanks are due to the course instructors (Dianne Newman, Jared
Leadbetter, Scott Dawson and Kurt Hanselmann) who helped troubleshoot the authors ideas and
suggested new paths of research. Finally, attendance at this course would not have been possible
without the generous scholarships granted by the Aline D. Gross Foundation and the Simons MD
Scholarship fund.
Citations
1.
Vitousek PM, Howarth RW (2007) Nitrogen Limitation on Land and in the Sea : How Can
It Occur ? Nitrogen limitation on land and in the sea : How can it occur ? 13:87–115. doi:
10.1007/BF00002772
2.
Saviola, Bishai (2006) The Prokaryotes.
3.
Tuschak C, Glaeser J, Overmann J (1999) Specific detection of green sulfur bacteria by in
situ hybridization with a fluorescently labeled oligonucleotide probe. Arch Microbiol
171:265–272. doi: 10.1007/s002030050709
4.
Yamaguchi T, Kawakami S, Hatamoto M, et al. (2015) In situ DNA-hybridization chain
reaction (HCR): A facilitated in situ HCR system for the detection of environmental
microorganisms. Environ Microbiol 17:2532–2541. doi: 10.1111/1462-2920.12745
5.
Ludwig W, Strunk O, Westram R, et al. (2004) ARB: A software environment for
sequence data. Nucleic Acids Res 32:1363–1371.
6.
Glöckner FO, Amann R, Alfreider A, et al. (1996) An In Situ Hybridization Protocol for
Detection and Identification of Planktonic Bacteria. Syst Appl Microbiol 19:403–406.
Figures:
Figure 1: Top left, 100x phase contrast image of I1 enrichment four days after leaving the headspace undisturbed. Top, mono-FISH image of I1 enrichment at the same time point. DAPI
staining is cyan, Chlorobi probe is green. Bottom, cultures at various time points after leaving the
head-space undisturbed. The five on the left have been undisturbed for a week the three on the
right have been undisturbed for 4 days, the far right bottle is an un-inoculated control.
Figure 2: qPCR measurements of Chlorobi-specific NifH genes across samples. Only
enrichments and the seawater table have appreciable amounts of the C-NifH gene. The color and
size of the points corresponds to the dilution factor in the qPCR plate. Similarly sized and
colored samples are comparable.
Figure 3: Gas chromatography measurements comparing the amount of acetylene to ethylene at
initial time points and after 4 hours of incubation. The area is on a log scale because of the
extremely high initial concentration of acetylene to ethylene. Using a paired t-test, only the
change in ethylene is significant (p = 0.0303). Boxes display the mean and standard error of the
measurements.
Figure 4: An example of filamentous microbe stained with the HCR-FISH general NifH probe.
In cyan, is general DAPI stain. Green indicates HCR NifH probe.