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Home , Oxidosqualene cyclase, Squalene, Tetrahymanol

Marisa Mayer

Microbial Diversity 2016

Utilizing Cyclase (STC) Degenerate Primer PCR and IMMUNO- FISH to Identify New Microbial in the Environment

BACKGROUND:

While there remains a large number of unknowns left to explore across the realm of modern microbiology, few areas remain quite as poorly explored as that of microbial eukaryotes in the environment. Microbial eukaryotes can serve as predators, benefactors, or symbiotic hosts for a number of and archaea. Most attention to microbial eukaryotes has been based on the medical field and the pathogenicity of certain microbial eukaryotes, while their distribution and biology in their native environments has been largely ignored. These large gaps in the distribution and function of microbial eukaryotes in different ecosystems need to be addressed in order to truly understand the dynamics of different environmental ecosystems.

An especially unexplored region of microbiology includes the study of anaerobic diversity and function in the environment. This lack of knowledge is partially driven by undersampling of microbial eukaryotes in anaerobic environments and partially driven by the lack of tools developed for identifying microbial eukaryotes in environmental samples. Through my mini project here at the MBL Microbial Diversity course, I hoped to apply degenerate primer PCR, used previously in bacterial environmental samples (Ricci et al., 2014), to identify novel anaerobic eukaryotes.

We wanted to use degenerate PCR primers to amplify all known diversity of the squalene tetrahymanol cyclase (stc) in our environmental samples. Stc is a gene that cyclizes squalene into tetrahymanol in such as (Mallory et al., 1963; Saar et al., 1991). These microbial eukaryote tetrahymanol producers use the tetrahymanol as an analogue for in their . Sterols cannot be produced in these organisms because the needed to cycle squalene into sterols, the (osc), cannot function in the absence of sufficient oxygen. Bacteria, also generally lacking sterols in their membranes, have also been found to produce tetrahymanol using a separate pathway (Banta et al., 2015). The bacterial pathway combines the hopanoid producing gene, the squalene hopene cyclase (shc), and a newly discovered gene, the tetrahymanol synthase (ths) gene, to generate tetrahymanol (see Figure 1). squalene shc osc

sterols

Figure 1: Three pathways for or tetrahymanol production in eukaryotes and bacteria. Modified from Banta et al. (2015).

Sterols are found in the membranes of organisms across almost all of the eukaryote tree, and thus they are thought to be essential. For this reason, it is no surprise that organisms living in oxygen conditions not conducive to sterol production would develop a pathway to generate a to take over the functions of sterols. Tetrahymanol is a triterpenoid, 5-ringed that does not require oxygen for its . Tetrahymanol is a particularly interesting molecule as its diagenetic product, gammacerane, is a biomarker molecule with implications for past environments in the rock record. Usually tetrahymanol serves as a proxy for oxygen stratification in past ecosystems. It is thought that gammacerane in the rock record stems from ciliates such as tetrahymena and paramecium living in anaerobic conditions who produced the tetrahymanol in place of sterols.

Biomarkers are a solution to the detection problem of microbes in the rock record (Eglinton et al., 1964; Eglinton and Calvin, 1967). Biomarkers are the organic molecular remains of living systems such as structurally preserved remains of and geochemical remnants of past life. Biomarkers studied range from sterols such as (Li et al., 1995), hopanoids such as bacteriohopanepolyols (Summons et al., 1999; Talbot et al., 2003; Waldbauer et al., 2009), or photopigments such as chlorophyll (Jeffrey et al., 1997). Usually in the form of deteriorated , biomarkers are commonly found as fossilized organic matter in petroleum (Ourisson & Albrecht, 1992). These complex, biologically produced are found in rocks that can date back to ~2.5 billion years ago (Brocks et al., 2003). Because of their durability and longevity, biomarker molecules can be used to detect life in ancient sediments dating back to the origins of life on Earth and possibly in deposits on other planetary bodies in the solar system (Simoneit et al., 1998; Summons et al., 2007; Allwood et al., 2013; Jahnke et al., 2014).

Many diagnostic remnants of past life can be discovered in ancient sediments and give clues to the nature of microbial life on the early Earth, but these clues are only as good as our interpretation of modern systems. When it comes to microbial eukaryotes, this understanding of their function in modern environments are not understood well enough to decipher the distribution of tetrahymanol in the environment.

By using degenerate PCR primers to amplify the squalene tetrahymanol synthase gene in our environmental samples of anaerobic sediments, I hoped to expand our knowledge of anaerobic eukaryotes in samples from Trunk River. Degenerate primers allow us to search for a gene of interest in organisms whose have not been sequenced. I also hoped that the stc primers would find more new sequences of stc than were previously known. The increased number of known anaerobic microbial eukaryotes could then inform a more refined tree of stc . This could perhaps help elucidate the evolutionary story of tetrahymanol synthesis in anaerobic eukaryotes.

GOALS AND HYPOTHESES:

Takishita et al. (2012) previously proposed a tree of the evolutionary relationships between bacterial and eukaryote stc, osc, and shc in which they proposed that the stc gene was laterally transferred from bacteria to the eukaryotes. This tree can be seen in Figure 2. We believe that these interpretations of stc and its evolutionary history are problematic given a) the sparse number of taxa used to construct their tree, b) that the taxa the authors did include in their tree are evolutionary distant from one another, and c) bacteria do not have an stc gene, but instead generate tetrahymanol through a separate pathway which combines the squalene hopene cyclase (shc) gene and the tetrahymanol synthase (ths) gene as described by Banta et al. (2015) and depicted in Figure 1.

The notion that key eukaryotic pathways such as the lipid biosynthesis pathways were acquired from horizontal gene transfer has remained in the literature for some time now (Keeling, 2009; Tomazic et al., 2014). In this model, the genes for these processes are not a part of the evolution of the eukaryotes, but were acquired separately from a transfer event somewhere along the line of evolutionary time. Not through vertical inheritance as normal genes are transferred. However, in this instance, it is hard to imagine and seemingly unparsimonious that such an essential gene that produces the only sterol functional equivalent in anaerobic organisms could have been acquired by a chance transfer. Even more hard to imagine, is how such evolutionarily distant eukaryotes could have all acquired this same gene transfer multiple times. These difficulties in conceptualization, in conjunction with the lack of taxa used to construct the Takishita et al. (2012) tree, are why we hypothesized that the squalene tetrahymanol cyclase gene (stc) arose as an early, novel pathway in anaerobic eukaryotes. Furthermore, we hypothesized that finding more anaerobic eukaryote stc sequences would help evolutionary trees of this gene reflect this relationship.

My goal for this mini project was to become comfortable with molecular techniques of environmental exploration. In particular, I wanted to learn the skills of degenerate primer construction, PCR methods of probing environmental sample DNA for genes of interest, and the theory behind constructing better phylogenies that can depict more accurate evolutionary relationships between groups of organisms for later hypothesis testing. I also was interested in adding to the amount of data out there on anaerobic eukaryotes as the current amount of knowledge is rather scarce.

Figure 2: A tree of lipid biosynthesis genes OSC, STC, and SHC and their proposed evolutionary relationships in bacteria and eukaryotes. From Takishita et al. (2012).

METHODS:

Environmental Sampling and DNA Extractions:

To collect low oxygen environmental samples, we took sediment cores in Trunk River Pond. Figure 3 illustrates the core intact with the various layers marked. We hoped to capture a gradient of oxygen concentrations from the bottom water column to the deep sediments. The cores were roughly 30 cm in depth.

water

organics

organics and sand

sand middle

Bottom organics and sand

Figure 3: Trunk River Pond sediment core sample with sample layers delineated. Before segmenting the core into various layers of interest for downstream analysis, oxygen concentrations were measured by microelectrode probe (see Figure 4). The oxygen concentrations were combined with visual color-based boundary zones in the core to determine the different depths at which sample distinctions should be separated into. The samples were then separated and prepared for DNA extraction of the 5 sediment layers: top oxygenated water, top decaying organics, mixed organics and sand, middle sand, and bottom mixed organics and sand.

Eukaryote DNA in the environmental samples was extracted using the MoBio Power Fecal DNA Isolation Kit. Two control DNA samples were also prepared to check for the stc and 18S primer effectiveness. The controls chosen include a pure culture paramecium species and a . These control samples were extracted using the Promega Wizard Genomic DNA Purification Kit.

Degenerate Primer Design:

Alignments for the gene of interest were based around the sequences from the taxa described by Takishita et al. (2012) to have homologue stc genes. Stc sequences were attained from NCBI GenBank using the Blast search function of genomes. We performed a ClustalX Muscle multiple sequence alignment in Jalview to align our stc sequence data. Avoiding higher degeneracy amino acids, we chose four regions on the stc gene sequence to create two forward degenerate primers and two reverse degenerate primers.

Gradient PCR Amplification:

We explored a number of primer combinations on our environmental samples at a range of temperatures (from 45ºC to 65ºC, 28 PCR cycles) to attempt to amplify stc genes in our sediment layer samples and control samples. Our degenerate primer sequences for stc are as follows:

Table 1: List of degenerate primers created to amplify the squalene tetrahymanol cyclase gene in environmental eukaryotes. DEGENERATE STC PRIMER BASE PAIR SEQUENCE STC 54F 5’[TGGNNNTAYCCNCCNTAYAARGG]3’ STC 128F 5’[TTYAAYTAYTKBKMNYTNAAR]3’ STC 590R 5’[YTAHATYTYYTTNARNCCCCA]3’ STC 710R 5’[YTANANNCCNSMYTGNCCNGTNCC]3’

All combinations of the four degenerate primers were attempted, as well as an 18S primer screen as a control to ensure that eukaryotes could be found in our DNA samples.

Microscopy:

We attempted to perform a tubulin immuno stain combined with fluorescence in situ hybridization (IMMUNO-FISH) of microbial eukaryotes in the environmental samples. The eukaryotes were stained with the immuno stain (Green fluorescence) and in some cases a eukaryote FISH probe, an alpha-proteobacteria FISH probe, a gamma/delta-proteobacteria FISH probe, or an archaeal FISH probe. The goal was to see which eukaryotes may be found in the various layers of the oxygen transition zone in the sediment. The alpha-proteobacteria, gamma- proteobacteria, and archaeal FISH probes were also utilized to look for symbionts of the microbial eukaryotes in the samples.

RESULTS:

The oxygen concentration data from the microelectrode probe show that oxygen concentrations begin to drop rapidly around the 2 cm mark. The oxygen concentrations at different depths in the core can be seen in Figure 4. The zero mark was decided to be where the air/water interface was at the top of our core sample. Oxygen Depth Profile

0

20000 Depth (µm)

40000

60000

0 100 200 Oxygen Concentration (µmol/L)

Figure 4: Plot of oxygen concentrations at the various depths of the Trunk River sediment core. Measurements taken by microelectrode probe. The degenerate primers and 18S primers were both successful in the first primer test PCR that we performed on our environmental samples. We tested the STC 54F and STC 590R degenerate primer pair first. We chose to test the organics and top mixed organics and sand layer first because we suspected that there would be more anaerobic eukaryotes here than the other layers. As can be seen in Figure 5, there were bands that appeared in both 18S primer PCR product and some bands in the STC primer PCR products. However, all subsequent STC primer tests failed to produce any visible bands. A variety of DNA concentrations, primer concentrations, PCR temperatures, and even a variety of DNA sample types were implemented to try and get the degenerate primers to produce a band on a 1% agarose gel. Even still, there was not enough time to get PCR conditions optimized to consistently produce bands with our stc primers such that I could be confident in the success of the primers I designed. 18S STC 18S organics STC organics organics and organics and sand sand

Figure 5: Image of PCR products from first degenerate and 18S primer test. Based on this result, we proceeded by running a PCR on all sample layers with 18S primers to get sequence data on which eukaryotes we had found in our Trunk River Samples. Unfortunately, the clone libraries when sent for sequencing failed and only vector sequence was returned in the data. As a result, we were unable to perform any analysis of eukaryote distributions in the sediment samples.

The IMMUNO-FISH samples were not successful upon first attempt. The probes all worked in terms of staining tubulin and marking DNA, yet there were simply not enough cells present to be stained to get a good sense of the eukaryote distribution and diversity in the various layers of our sample. There was also a lot of autofluorescence in the samples in the first attempt. Perhaps this could be attributed to the large amount of chromatium species that others working in Trunk River had observed already.

However, a second attempt was made at immuno staining to see if the tubulin immuno stain was working on eukaryotes in environmental samples at all. In this second attempt it was necessary to omit the FISH probe segment of the protocol due solely to time constraints of the course. This second attempt was performed with 10 ml of sample instead of our initial 100 µl worth of sample. As a result of the higher cell densities, the protocol worked and a number of diverse eukaryotes were identified in our microscopy work (Figure 6). Our time constraints also prevented us from collecting a new sediment core from Trunk River to run the immuno stain on. Therefore, we used the seawater table enrichment that was readily available in the lab to test the methodology.

In future work, we would like to apply the IMMUNO-FISH technique to Trunk River environmental samples to observe the eukaryotes in that environment as well. There were a number of ciliates, flagellates, and other strange morphologies found in this second attempt (see Figure 6 for examples). This suggests that this technique can be useful for future explorations of eukaryotes in new environmental samples.

Figure 6: The diversity of eukaryote morphologies in environmental samples. The eukaryotes are stained green due to the tubulin immuno stain while the bacterial DNA and eukaryotic nucleus are DAPI stained blue.

DISCUSSION:

The sheer number of empty gels in our degenerate PCR tests suggest that there is a lot of work left to do in optimizing our methods to successfully identify new squalene tetrahymanol cyclase in the environment. We could not get bands to appear for primer tests run on control samples of DNA extracted from two different ciliate isolates. This could be due to a number of reasons. The primers could have failed because the PCR conditions were not correct for this primer to bind to our sample DNA, or because our primers were not specific enough to amplify the gene of interest, or because the DNA quality was not high enough, or even because the sample DNA did not contain the stc gene in the first place. These ciliate isolates had no sequence data to check for the presence or absence of stc. In future work, having a control that confidently possesses the stc gene would be helpful in determining the quality of our degenerate primers and the success of our PCR.

These failed PCRs were useful in of themselves though. Not getting the primers to produce bands forced me to really consider the possible problems that could arise in our PCR reaction design. I had to learn how to logic through which parts of our reaction can be tweaked to try and get results in a systematic, experimental manor. I never reached a conclusion for why our PCR experiments failed, but I feel that given more time, a working primer set could have been constructed.

Our immuno stain results were more successful for identifying new eukaryotes. Unfortunately, they cannot point to the presence or absence of tetrahymanol. But, in future, it is not inconceivable that FISH probes could be designed to incorporate our stc primers to tag for that gene once the primers are optimized. The alpha-, gamma-, archaeal, and eukaryote FISH probes worked successfully, but there was not enough sample to get a good survey of the diversity in the samples. In future work with sufficient starting sample concentrations there is no reason that the FISH component could not be reintroduced to look for bacterial symbionts or other interesting interactions between the eukaryotes, bacteria, and archaea in environmental samples.

REFERENCES:

Allwood, a C., Burch, I. W., Rouchy, J. M., & Coleman, M. (2013). Morphological biosignatures in gypsum: diverse formation processes of Messinian (∼6.0 Ma) gypsum stromatolites. Astrobiology, 13(9), 870–86. http://doi.org/10.1089/ast.2013.1021

Banta, A. B., Wei, J. H., & Welander, P. V. (2015). A distinct pathway for tetrahymanol synthesis in bacteria. Proceedings of the National Academy of Sciences of the United States of America. http://doi.org/10.1073/pnas.1511482112

Brocks, J. J., Buick, R., Summons, R. E., & Logan, G. A. (2003). A reconstruction of Archean biological diversity based on molecular fossils from the 2.78 to 2.45 billion-year-old Mount Bruce Supergroup, Hamersley Basin, Western Australia. Geochimica et Cosmochimica Acta, 67(22), 4321–4335. http://doi.org/10.1016/S0016-7037(03)00209-6

Eglinton G, Scott PM, Belsky T, Burlingame AL, Calvin M. Hydrocarbons of Biological Origin from a One-Billion-Year-Old Sediment. Science. 1964 Jul 17;145(3629):263–264.

Eglinton, G., & Calvin, M. (1967). Chemical fossils. Scientific American, 216, 32-43.

Jahnke, L. L., Turk-Kubo, K. A., N. Parenteau, M., Green, S. J., Kubo, M. D.Y., Vogel, M., Summons, R. E. and Des Marais, D. J. (2014), Molecular and lipid biomarker analysis of a gypsum-hosted endoevaporitic microbial community. Geobiology, 12: 62–82. doi:10.1111/gbi.12068

Jeffrey, S.W., Mantoura, R.F.C., & Wright, S.W. (1997). Phytoplankton pigments in oceanography: guidelines to modern methods. 661p. Paris: Unesco Publishing, Journal of the Marine Biological Association of the United Kingdom, 77(03), 918. http://doi.org/10.1017/S0025315400036389

Keeling, P. J. (2009). Functional and ecological impacts of horizontal gene transfer in eukaryotes. Current Opinion in Genetics & Development, 19(6), 613–619. http://doi.org/10.1016/j.gde.2009.10.001

Li, W., Dagaut, J., & Saliot, A. (1995). The application of sterol biomarkers to the study of the sources of particulate organic matter in the Solo River system and and Serayu River, Java, Indonesia. Biogeochemistry, 31(3), 139–154. http://doi.org/10.1007/BF00004046 Mallory, F. B., Gordon, J. T., & Conner, R. L. (1963). The isolation of a pentacyclic triterpenoid alcohol from a protozoan. Journal of the American Chemical Society, 85(9), 1362-1363.

Ourisson, G., & Albrecht, P. (1992). Hopanoids. 1, Geohopanoids: the most abundant natural products on Earth? Accounts of Chemical Research, 25(3), 398–402. http://doi.org/DOI: 10.1021/ar00021a003

Ricci, J. N., Coleman, M. L., Welander, P. V, Sessions, A. L., Summons, R. E., Spear, J. R., & Newman, D. K. (2014). Diverse capacity for 2-methylhopanoid production correlates with a specific ecological niche. The ISME Journal, 8(3), 675–84. http://doi.org/10.1038/ismej.2013.191

Saar, J., Kader, J.-C., Poralla, K., & Ourisson, G. (1991). Purification and some properties of the squalene-tetrahymanol cyclase from Tetrahymena thermophila. Biochimica et Biophysica Acta (BBA) - General Subjects, 1075(1), 93–101. http://doi.org/10.1016/0304- 4165(91)90080-Z

Simoneit, B. R., Summons, R. E., & Jahnke, L. L. (1998). Biomarkers as tracers for life on early earth and Mars. Origins of Life and Evolution of the Biosphere : The Journal of the International Society for the Study of the Origin of Life, 28(4-6), 475–83. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9776659

Summons, R. E., Jahnke, L. L., Hope, J. M., & Logan, G. A. (1999). 2-Methylhopanoids as biomarkers for cyanobacterial oxygenic photosynthesis. Nature, 400(6744), 554–7. http://doi.org/10.1038/23005

Summons, R. E., Albrecht, P., McDonald, G., & Moldowan, J. M. (2007). Molecular Biosignatures. Space Science Reviews, 135(1-4), 133–159. http://doi.org/10.1007/s11214- 007-9256-5

Takishita, K., Chikaraishi, Y., Leger, M. M., Kim, E., Yabuki, A., Ohkouchi, N., & Roger, A. J. (2012). Lateral transfer of tetrahymanol-synthesizing genes has allowed multiple diverse eukaryote lineages to independently adapt to environments without oxygen. Biology Direct, 7, 5. http://doi.org/10.1186/1745-6150-7-5

Talbot, H. M., Summons, R., Jahnke, L., & Farrimond, P. (2003). Characteristic fragmentation of bacteriohopanepolyols during atmospheric pressure chemical ionisation liquid / trap . Rapid Communications in Mass Spectrometry : RCM, 17(24), 2788–96. http://doi.org/10.1002/rcm.1265

Tomazic, M. L., Poklepovich, T. J., Nudel, C. B., & Nusblat, A. D. (2014). Incomplete sterols and hopanoids pathways in ciliates: Gene loss and acquisition during evolution as a source of biosynthetic genes. Molecular Phylogenetics and Evolution, 74, 122–134. http://doi.org/10.1016/j.ympev.2014.01.026 Waldbauer, J. R., Sherman, L. S., Sumner, D. Y., & Summons, R. E. (2009). Late Archean molecular fossils from the Transvaal Supergroup record the antiquity of microbial diversity and aerobiosis. Precambrian Research, 169(1-4), 28–47. http://doi.org/10.1016/j.precamres.2008.10.011