July 31, 2007
Ana Gutiérrez-Preciado
Instituto de Biotecnología, Universidad Nacional Autónoma de México
Microbial Diversity Course
Marine Biological Laboratories, Woods Hole, MA
Isolation of chemotactic sulfate reducing bacteria.
Abstract
Different enrichments are used to isolate sulfate reducers using H2, lactate,
butaryte and ethanol as electron donors. Pure cultures were never obtained.
Sulfate reducers were always obtain as well as acetogenes. This could be
because the sulfate reducers that are being isolated need acetate to reduce
sulfate. Chemotaxis Assays were used in order to get insights of which electron
donors and electron acceptor are preferred by the isolated sulfate reducers.
Finally, enrichment for iron reducers was made and inoculated with our sulfate
reducers culture. Growth was present in the iron media, but whether these
organisms are iron reducers remains to be determined.
Introduction
Sulfate is the most oxidized form of Sulfur, is one of the major anions in water and is used by sulfate reducing bacteria a group that is phylogenetically widespread. The end product of sulfate reduction is H2S (1). Many organisms (plants, algae, fungi and most bacteria) use assimilative sulfate
reduction to form sulfide to get sulfur as a source for biosynthesis (i.e.,
synthesize amino acids). The ability to use sulfate as an electron acceptor for
energy-generating processes, however, involves the large-scale reduction of
2- SO4 , which is restricted to sulfate reducers. In dissimilative sulfate reduction,
sulfide is excreted (1).
H2, lactate, and pyruvate are widely used as electron donors by a variety of sulfate reducers, whilst other electron donors, such as, ethanol (and other alcohols), fumarate, malate, choline, acetate, propionate, butyrate, long-chain fatty acids, benzoate, indole and hexadecane have more restricted use (1).
(Figure taken from (1)) Due to the high concentration of sulfate in seawater, sulfate reducers play a
dominant role in the complete degradation of organic matter in anaerobic marine
systems. Phylogenetically, the sulfate reducers are found among the δ
Proteobacteria (Desulfovibrio spp.), Archaea (Archeoglobus spp.) and
Clostridium-Bacillus subphylum (Desulfotomaculum spp.).
Questions:
2- 2- 0 1. What are the bacteria sensing? SO4 ? SO3 ? S2O3? S ? H2S?
2. Are these compounds chemoattractant? Or chemorepelent?
3. Which electron donors are chemoattractant? Lactate? H2? Butyrate?
Ethanol? Acetate?
4. If I get any sulfate reducers…
4.1. Are they also Iron reducers?
Sampling
For the enrichments we already have of sulfate reducers (Group 4), a sample was taken from Trunk River on Jun 19th, 2007, in the deep sediments.
Enrichment
The sample that was taken contained ~15 cm of water and ~15 of sediments in
depth. Samples from the sediment were taken to look for anaerobic organisms.
The enrichments were settled with marine water and with two electron donors: H2 and methanol.
Six enrichments were done with seawater media with:
1. H2 + CO2
2. H2 + CO2 +BES
3. H2 + CO2 +Na2SO4
4. MeOH
5. MeOH + BES
6. MeOH + Na2SO4
Enrichments will be monitored periodically for the production of methane (by Gas
Chromatography) and acetate (via HPLC). The production of sulfide will be detected by a chemical assay or by Hydrogen Sulfide Microsensors (Unisense). Basal anaerobic modular seawater medium (see lab manual Microbial Diversity
2007).
Only the 3rd enrichment was used, because it is enriching for sulfate reducers.
Isolation of pure cultures
Isolation of these organisms was done by two approaches. First, plating in Agar plates with the media described above. As a second approach, we employed plating the 3rd media on a solid surface. Silica plates were chosen since agar is not useful substrate for direct plating of marine samples (because of the prevalence of agar-degrading organisms). These plates were incubated under strictly anaerobic conditions in a custom built anaerobic incubator at 30°C.
Characterization of isolated organisms
Characterization will be done in three ways:
1. 12 isolated bacterial colonies will be chosen from the silica plates. 16S
rRNA sequences will be determined via PCR amplification (2) and
sequencing. Phylogenetic relationships will be determined with ARB (3).
2. Different substrates will be provided to the bacteria to see which the best
for its growth is. Test for elector donors, such as Lactate, Butyrate and
Ethanol will be made.
3. Chemotaxis Assays (described below)
Chemotaxis Assays
In chemotaxis assays, glass capillaries are loaded with defined substrate
solutions, inserted in a suspension of motile microorganisms, and the
accumulation of cells at the opening of or within the capillary is monitored by
direct or indirect methods (4). This new modified method described by Overmann
J (see ref (4)) was used for parallel testing of a large number of different
chemoattractants within the same bacterial sample.
Flat rectangular glass capillaries with a length of 50 mm, an inside diameter of
0.1 x 1.0 mm, and a capacity of 5 µl (Vitrocom, Mountain Lakes, NJ) were used
for looking at the microscope. These capillaries fit exactly into the opening of the
chemotaxis chamber (that consists of a bottle that can be filled with 20 ml of the
sample and is closed with plasticine for anoxic incubations). The specific
geometry of these capillaries permits a direct light microscopic examination of
their contents (4).
In this case, glass capillaries will be loaded with the propionate, butyrate, lacatate
2- and ethanol as electron donors. Electron acceptors, such as sulfate (SO4 ), were also tested. Additionally, elemental sulfur (S0) as well as taurine, cysteine and
thiosulfate were tested. Stock solutions of these test substrates were prepared at
a concentration of 100 mM. These solutions were sterilized by filtering them in
the anaerobic chamber. Posterior dilutions were made in order to have the
chemoattractants at a final concentration of 1 mM. Capillaries are filled with
diluted test substances by capillary action, sealed at one end with plasticine
(Idena, Berlin, Germany), and inserted directly into the microscopic chamber so that their open ends extend well into the sample. This creates a gradient of the
tested compound. At least two parallels will be used per substrate, one flat
capillary for direct. Each chemotaxis assay also comprises two control capillaries,
which are filled with sterile culture supernatant or sample water. This technique permits a rapid screening of a limited number of chemoattractants. For more details on this technique, see ref (4).
Results
12 colonies were picked from silica and agar plates and transfer into sea water based medium with NaSO4 and a head space of H2/CO2.
silica plate agar plate
Only one tube grew from a colony I picked from Group 3 agar plate. The original sample was taken from Trunk River. The “pure” culture was observed at the microscope revealing two cell types:
HPLC analysis revealed that they were producing acetate:
The 15.537 peak indicates acetate presence at a 24.551 mM concentration. This indicates presence of acetogenes in the culture. The 14.310 peak indicates formate presence. This makes sense because formate is an intermediate of the acetyl CoA pathway. The concentration of the formate was not determined.
Cells were lysed by the freeze thaw method and PCR amplification of the 16 S rRNA region was done using the 8F and 1492R primers (5). Since different cell types were observed, a clone library was made out of this DNA using the invitrogen TOPO TA cloning kit. Sequencing was done at the Mitch Sogin lab.
Returned sequences reveal that no sulfate reducers were present in this culture, but rather only acetogenes were found. This tree was constructed by Maximum Likelihood using the ARB software (3).
These five sequences belong to three different acetogenes, as revealed by neighbor joining similarity matrices using the ARB software (3):
5 A06 D06 1.000000 E05 0.983968 0.982533 E06 0.998127 0.998148 0.985915 H06 0.984252 0.982906 1.000000 0.986193
D06 is equal to A06, and H06 is equal to E06.
No chemotaxis assay was performed with this culture
Since there was no detectable growth on the “pure” culture tubes, I sampled again Trunk River and Sippewissett.
Trunk River:
Sippewissett:
Chemotaxis assays were performed using the first two environmental samples from Trunk River and the first one from Sippewissett.
The capillary tubes
were use to extract the
cells and lyse them
using the freeze thaw
protocol for further 16S
rRNA PCR
amplification (5).
Micrographs from Chemotactic Assay on Trunk River environmental sample:
Control capillary tube Taurine capillary tube
Cyano present in ALL capillary Sulphur capillary tube tubes
Enrichments
The Basal anaerobic modular seawater medium was slightly modified. Ferrous sulfate was used instead of sodium sulfate. The only reducing agent used was cysteine, and it was added doubling the amount described in the lab manual
Microbial Diversity 2007. Three different media of this kind was made, by varying the electron donors: lactate, butyrate and ethanol.
Ferrous sulfate changes colour when sulfate is reduced to sulfide. So we chose this because colour will indicate us growth. The problem we found was that some environmental samples already contained sulfide, changing the color of the enrichment immediately after inoculation: Since tubes are incubated horizontally (to increase the surface of contact with the
H2/CO2 headspace), tubes were vigorously shaking and let them precipitate for half an hour and then look for growth:
From left to right: 1) the only tube with growth 2) a tube that has sulfide but no growth. 3) The control tube.
DNA was purified from these enrichments using the UltraClean Soil DNA Isolation
Kit from Mo Bio laboratories (http://www.mobio.com/support/protocols.php). 16S rRNA PCR amplification was done (5).
From all the PCR products obtained (from the capillary tubes and from the enrichments), clone libraries were made using the invitrogen TOPO TA cloning kit.
PCR amplification of the cloned sequenced in the vector was done using the M13 primers (described in the invitrogen TOPO TA cloning kit user manual). 28 PCR products were obtained: 2 3 3 2 2 2 2 F03 C0 D0 B03 E03 D0 F02 A03 A0 B0 C0 G0 E02 H02 C- 5 6 3 4 7 4 6 6 5 7 4 5 5 5 7 4 4 3 4 F06 F04 F05 A0 B06 D06 E06 A05 G0 C0 G0 A07 D0 G0 A04 C0 B0 C0 E0 H0 B0 C0 B0 D0 E0 D05 H06 G0 H0 H0 9 9 8 9 9 7 8 9 8 B10 F09 G0 H0 A10 A0 H0 F08 E09 B09 D0 C0 C08 D08 E07 H07 B08 F07 G0 A0 G0 E08 1 2 2 2 2 1 1 2 0 0 1 1 E1 G1 D1 F12 H1 G1 F11 D1 H1 A12 B12 C1 C10 F10 H10 D1 G1 A1 B11 E11 E10 C1
The 28 PCR products were ExoSAPped using the ExoSAP-IT kit from usb corporation (Cleveland, Ohio). Sequencing was performed by the Mitch Sogin lab.
26 sequences returned. From Group 3 enrichment chemotaxis assay:
This tree was constructed using Maximum Likelihood with the ARB software (3).
D12, F03 and E12 fall into the Bacteroidetes clade D12 and E12 were extracted from the lactate capillary tube, and F03 was extracted from the lactate capillary tube. C02 falls in the Mollicutes clade and it was extracted from the butyrate capillary tube. Is worth mentioning that all of the so-far described clades, are pathogens. C12 and D02 fall in the acetogenes clade and were extracted from the lactate and butyrate capillary tubes respectively. The remaining organisms are sulfate reducers from the δ Proteobacteria, Desulfovibrio spp. B08 and D08 were found in the Control capillary tube. H03 was extracted from the butyrate capillary tube, whilst C11, G12 and D11 were found in the lactate capillary tube.
Since Desulfovibrio spp. were found also in the capillary tube I am not able to conclude that they are attracted to lactate or butyrate.
From Group 4 enrichment chemotaxis assay:
This tree was constructed using Maximum Likelihood with the ARB software (3).
First of all, no PCR products were obtained from the capillary tube, so no conclusions can be made of whether which organisms are chemoattracted to which compound. B10 and E04 fall on the acetogenes clade and were found in the taurine and thiosulfate capillaries respectively. A03 is a Mollicute and was found in the Thiosulfate capillary tube. B03 is presumably a Desulfovibrio and it was also found in the thiosulfate capillary tube. B07 and G11 are Shewanella. This species can convert soluble metals and compounds, like uranium, into
insoluble forms. They are very versatile and can live aerobically, in the presence
of oxygen, or anaerobically, without oxygen. They can grow naturally almost
anywhere and does not cause disease in humans, animals or other organisms.
Iron is the principal electron acceptor for Shewanella and other metal-reducing bacteria thriving in environments lacking oxygen. Before bacteria and plants produced oxygen via photosynthesis, iron was likely the most abundant electron acceptor on prehistoric earth. Hence, metal-reducing organisms, like Shewanella, were likely to have developed before other respiratory organisms. G11 was found in the thiosulfate capillary tube, and B07 was found either in the S0 or in the cysteine capillary tube. (Tubes were accidentally dropped in the ethanol while lysing the cells erasing the label of these two tubes).
No direct counting was done, but it was clear (from microscope observations, and from number of tubes grown) that cells prefer lactate as an electron donor, then butyrate and at the end ethanol.
The following morphological cell types were observed in all the enrichments as the most common cell types:
Whether the first two cell types are the same
or are different was not determined.
A17 (Big brown colony from group 3 Agar plate S/H2CO2):
The white dots in the two right-most images appear to be sulfur globules. Some of them are free and others are probably inside the bacteria.
Trunk river sample enriched in media supplied with FeSO4 with butyrate as an electron donor:
The right-most picture show 3 bacteria attaches to what probably is a sulfur granule.
This is a cyanobacteria; the rightmost picture is
looked through the Filter 6 of the fluorescence
microscope.
Trunk river sample enriched in media supplied with FeSO4 with lactate as an
electron donor:
As mentioned above, two or three cell types were present in all my cultures. I
failed to get any pure culture. The few sequences I could get indicate that sulfate
reducers as well as acetogenes are present in all my cultures. Maybe the only
sulfate reducers I could “isolate” depend on acetogenes to grow, since some
sulfate reducers (heterotrophic) need acetate to reduce sulfate:
- 2- + CH3COO + SO4 + 3H →2CO2 + H2S + 2H2O
In a future attempt to growth them; acetate should be tested as an electron donor.
Iron reducers
In order to try if my sulfate reducers are also iron reducers, a new basal anaerobic modular seawater medium was tested. Sulfate was removed from the media, and instead amorphous iron was added as an electron acceptor. Electron donors were conserved: Lactate, Butyrate, and Ethanol. Previously described enrichments were used to inoculate these new media:
Due to time constrains, I couldn’t test if iron was being reduce and who was there. But my tubes were growing:
Control Control
Acknowledgments
Very special thanks to:
Mike and Bill for all their help, for teaching me so much about the anaerobic world
Jared, Bill and Tom, for insightful discussions on my project
Tracy and Kristen, for all our attempts to get some sequences
Rachel, for helping me set the chemotaxis assays
Stephanie for the coffee and for ARB. Dagmar for ARB
Reference List
1. Madigan, M. T. and J. M. Martinko. 2006. Metabolic Diversity. Sulfate Reduction, p. 560-563. In G. Carlson (ed.), Brock Biology of Microorganisms. Pearson Prentice Hall, United States of America.
2. Wilson, K. H., R. B. Blitchington, and R. C. Greene. 1990. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J.Clin.Microbiol. 28:1942-1946.
3. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Forster, I. Brettske, S. Gerber, A. W. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. Konig, T. Liss, R. Lussmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N. Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode, and K. H. Schleifer. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32:1363-1371.
4. Overmann, J. 2005. Chemotaxis and behavioral physiology of not-yet-cultivated microbes. Methods Enzymol. 397:133-147.
5. Wilson, K. H., R. B. Blitchington, and R. C. Greene. 1990. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J.Clin.Microbiol. 28:1942-1946.