So for the last couple of years I’ve supervised a series of undergraduates who have spent some time isolating bacteria from seagrass samples… sometimes from the plants themselves and sometimes from associated sediment. We usually used non-specific aerobic media such as Marine Broth and Seawater Nutrient Agar. The result has been a series of the usual suspects; Vibrio, Shewanella, Pseudoalteromonas, etc. We’ve sequenced a number of these genomes, examples of Genome Announcements papers like this can be found here, here, here, here, here,here and here. The goals of this culturing were three-fold; cool undergraduate projects (check), add seagrass-associated genome data to the database to aid in metagenomics and such (check), and to characterize isolates that might be important in seagrass biology (unknown).
In regards to the last goal, we’ve attempted to use abundance of taxa as one rough proxy of “importance” and significant changes within an experiment as another. We have several large 16S projects to work with, but in most cases the isolates that we have generated are not found at particular abundance or significance in these datasets. And perhaps that’s not surprising, non-specific media is good at isolating widely distributed generalists.
So now we plan to approach the problem from the opposite direction, we’re picking the OTUs from our 16S data that are the most interesting and attempting to selectively culture them. #1 on our hit list is Sulfurimonas which crops up over and over again.
Recently the Seagrass Microbiome group has been wrapped up in sending (and receiving!) microbiome sampling kits. These kits are part of a larger collaborative project focused on re-sequencing of Zostera marina samples in conjunction with sequencing of additional marine and freshwater Alismatid species and their microbiomes. JGI recently sequenced and released the Zostera marina genome, and we are hoping to build on their efforts and explore population level variation within Zostera marina, as well as differences in genome content and structure between Zostera and other Alismatids, in conjunction with microbiome sequencing.
The sampling kits sent by the seagrass microbiome group have focused on the microbial aspect of this project. We have asked members of the Zostera Experimental Network (ZEN) as well as additional collaborators to sample both plant tissue for sequencing (coordinated through Jay Stachowicz and Jeanine Olsen) and microbiome samples. We are extremely excited about this sample set, as it covers populations of Zostera marina across many different environments, for which we already have extensive metadata through the ZEN group! We are requesting root, sediment (within the rhizosphere), and leaf tissue, as detailed in the diagram below (courtesy of Jeanine Olsen).
Collaborators are also sampling at two depths per site (deep and shallow), so that we can examine microbiome differences that may correlate with population depth. We are sampling 24 individuals per site, 12 per depth.
So – am participating in a workshop, supported by CIFAR and the Gordon and Betty Moore Foundation over the next few days on “Marine Algal and Plant Microbiomes”. The workshop is basically trying to come up with a white paper / position paper on the future of such studies and to continue the conversation about this topic afterwards. We are asking questions like
What are the challenges and opportunities in this area?
What are the major scientific questions?
How are such systems different from fresh water or terrestrial systems?
How are they different?
How are marine systems involving other hosts (e.g., coral, sponges, dolphins) comparable (i.e., is there something about marine systems that links them together in any way).
What tools and resources could help advance work in this area?
So I am posting here asking for a few bits of information from any readers.
Are you interested in participating in follow up discussions on this topic?
Do you know of any people we should try to bring into the conversation even if they are not, well, you?
Are there any major projects in this area that would be worth engaging?
Any thoughts (on the topic that is) would be welcome.
My name is Karley Lujan and I am an undergraduate working on culturing bacterial isolates from the Seagrass microbiome. I joined this project because I am interested in learning about what information we can obtain from studying microbiomes. I think it is fascinating that although we can’t see microorganisms they are extremely prevalent and can have crucial roles in biological systems. The focus at the beginning of this project was to take Seagrass samples from Bodega Bay, create culture samples, and use Sanger sequencing of the 16S rRNA to identify what we grew. Seagrass and sediment samples were taken from Bodega Bay, CA. Then, in order to obtain isolates from the seagrass, we focused on the leaves, roots, and sediment. What we were able to successfully extract DNA from were identified as Shewanella, Pseudoalteromonas, Colwellia, Tenacibaculum, Vibrio and Alteromonas.
Sample preparation: Dilutions of sediment with PBS, PBS rinse of roots and leaves, ground and crushed leaves with PBS
Culturing: Plated the PBS sample solutions onto two of each of the following plate types; one plate for 25℃ and the other at 4℃
Agar Plates/Liquid Media
Difco Marine Broth
Selected Colonies: After there was significant growth on the plates we selected various interesting colonies and isolated them by dilution streaking. Single colonies were then grown overnight in the appropriate liquid media and at the appropriate temperature
DNA Extraction: Genomic DNA extractions were performed and glycerol stocks were made using the successful liquid cultures. Extracted DNA then went through 16S rRNA gene PCR and gel electrophoresis in order to confirm that enough DNA was present for Sanger Sequencing
Sanger Sequencing: 16S rRNA sequences for each isolate were ran through BLAST and phylogenetic trees were built in order to obtain tentative identifications for the isolates
Results: After Sanger sequencing, the data was ran through BLAST to obtain a tentative identification and determine whether or not the microbe was a good candidate for sequencing.
Shewanella:Electrogenic- An electron generator that can be used in microbial fuel cells.
Vibrio:Some species of Vibrio can go through morphogenetic changes after going from a liquid to a solid surface. This leads them to change from swimmer cells to swarmer cells.
Pseudomonas:Two bacterial isolates were cable of growing on Nitrogen-Free agar plates at 25⁰C. Identified as part of the genera Pseudomonas, there are some species of this genera capable of aerobically fixing nitrogen. These are of particular interest as we will be further investigating which nitrogen-fixing bacteria are essential for seagrass health.
Currently I am beginning to look at the genomes of the bacteria we decided to sequence and I am also working with bacteria that are capable of growing on the nitrogen-free agar plates. At first it was difficult to extract the DNA from these bacteria but now both have been tentatively identified as Pseudomonas through sanger sequencing of the 16S gene. This is interesting because there aren’t many Pseudomonas that can fix nitrogen which is what these two must be doing in order to survive on the nitrogen-free plates. These two bacteria also have different morphologies which means they could be different species in the genus Pseudomonas. Due to their morphological similarities yet ability to grow on nitrogen-free agar, I think these two bacteria are very interesting and we will be finding out more about them by sequencing and analyzing their genomes.
A paper on the genome of Z. marina was released early this year. This is the first marine angiosperm genome to be sequenced, and since it’s our main host organism, we are fairly excited. This opens up a couple research possibilities, like studying host-microbe coevolution between Z. marina and its microbiome. We can also use the plant reference genome with RNA-seq data to filter out reads from the microbes, which will make it easier to look at the gene functions represented in the seagrass microbiome. Having a reference genome for our host will definitely come in handy, and has opened some exciting new doors for us.
Some of us at team Seagrass participated in a workshop on SEM (scanning electron microscopy) sponsored by the Electron Microscopy Lab at UC Davis. For this week only Hitachi Tabletop SEM was made available to researchers to test out. SEM is a great tool to produce high resolution images, especially for samples for which preparation techniques would otherwise alter the sample. We decided this would be a great opportunity to visually explore microbial diversity on seagrass roots/leaves/rhizomes. The amount of sample and preparation is minimal relative to other techniques. We’ve been particularly concerned with our previous FISH images, as FISH requires many washes, and may be removing microbes from the surface of our samples.
We put a minimal amount of sample of root (top), fresh leaf (right), decaying leaf (bottom), and rhizome cross section (left) on a piece of carbon conductive paper (sticky on both sides), which was stuck on top of the SEM sample mount. We then inserted the mount into the SEM and turned on the vacuum. The vacuum for this particular SEM is actually a partial vacuum, which maintains a small amount of air molecules within the chamber, which produces a better image in the absence of coating your sample with conductors.
The results were great! We were able to see a lot of diatoms spread out across the leaf surface, as well as some interesting plaque (?) formations on the root tips. We also noticed differences in diatom abundance between the live and decaying leaves, with the live leaf being completely covered with diatoms. Diatoms are marine microbial eukaryotes (Heterokonts) that form silica based outer layers, often resembling complex geometric patterns. Diatom assemblages have previously been characterized in Thalassia testudinum and in Zostera marina. We’re excited to use the SEM to explore microbial diversity on additional seagrass species and freshwater relatives within the Alismatales. Hopefully these results can help inform our future culturing experiments.
A few of us at Team Seagrass have been doing fieldwork in Northern CA in order to collect seagrass relatives. We have specifically been targeting freshwater species (highlighted in blue in the below phylogeny). We will continue with marine and brackish species when the tides in the San Francisco Bay are lower.
For further plant identification and the construction of a ‘host phylogeny’ we will be using chloroplast DNA markers.
In addition to what we have sampled thus far, we have also managed to sample some outgroups (Myriophyllum, Ceratophyllum, and Camboba). We will hopefully be able to sample additional outgroup species shown on the above tree.
We have been successful in 7 out of the 10 locations that we have tried. The lack of success so far can be attributed to drought conditions in CA and the increase in invasive submerged aquatic vegetation (e.g. Myriophyllum, Water Milfoil; and Eichhornia crassipes, Water Hyacinth). Water Milfoil and Water Hyacinth form dense mats below and above water, respectively, outcompeting native vegetation. Even in sites where we been successful, much of the area we surveyed has been overrun with these two invasive species.
Obviously drought has also been an issue for us, as many of the lakes and tributaries have little to no water in them this year, resulting in a lack of any aquatic vegetation. Water levels in Folsom Lake, for example, are so far reduced that the Park Service has had to build additional parking lots on the water side of the boat ramp in order for people to be able to get close to the new shoreline.
In the following weeks we will head up to the foothills in search of remaining freshwater species, as well as explore the Bay Area coastline and salt marsh/Delta area for marine and brackish species. Stay tuned!
During ZEN DNA extractions we noticed that samples preserved in the Zymo buffer were forming a precipitate with the C1 solution from the MoBio kit. Furthermore, many of the samples also resulted in very low DNA yields, perhaps correlated with the precipitate formation. Any microbiologist will tell you that there are many different ways to preserve samples from the field, but there does not seem to be a universal *best* method.
We decided the best way to approach this problem for the Seagrass Microbiome project was to explore a variety of sample preservation methods and see which approximated the ‘real’ microbiome best (as measured by preservation on dry ice). We chose the following methods to try: Dry ice, Zymo, RNA-later, Drierite, and Ethanol. We also wanted to investigate how different preservation methods performed over time. We chose 4 time points post sampling for our extractions: 24 hours, 1 week, 2 weeks, and 1 month.
We drove to Putah Creek in Winters in search of submerged aquatic plants in the Alismatales (the order that contains the seagrasses). Here is a photo of our study site:
We found a bed of Elodea canadensis growing near the shore and started sampling.
We pulled out whole plants and divided them into root and leaf for each of our trial preservation methods, with 3 replicates per method per time point. We extracted DNA at each of the different time points, and did PCR of the 16S SSU rRNA gene, with subsequent library preparation and sequencing on the Illumina Mi-Seq. We used QIIME to analyze the sequence data and compare species diversity between our preservation methods.
Our PCoA plot shows that preservation in Zymo best approximates the community captured using dry ice (our control), and that Drierite and Ethanol were both very different from anything else. RNA-later appears close to Zymo and dry ice, but we only have 5 data points for it. For the RNA-later and Ethanol extractions, we extracted directly from the solution without pelleting, which may have affected our end result.
The taxonomy table supports the trend seen in our PCoA plot, with Drierite and Ethanol being very different, and Zymo, dry ice, and our 5 RNA-later time points showing similar communities.
Looking at variation in community structure across time points within Zymo we see little to no change.
However, we see some change in community structure by the month mark with dry ice.
Take home messages from our sample preservation experiment:
Using Drierite as a preservation technique does not capture the community assembly well.
RNA-later and Ethanol could have had better success with pelleting and removing the supernatant prior to extraction. We may investigate this further in the future.
In spite of our initial concerns regarding precipitate forming in the Zymo buffer, Zymo is the clear winner in our trial.
There are slight changes in the community assembly by the month mark, in all methods (depending on whether you use weighted or unweighted unifrac PCoA).
Due to the current ease of sampling using dry ice, we will continue this method except in situations where samples must be kept at room temperature (we will then use Zymo).
While at the course, I also cultured a handful of microbes from seagrass leafs/roots and several different amoeba from seagrass bed sediments including one really cool “follow the leader” snail slime like amoeba (slime mold?).
Recently I’ve needed to visualize spatial changes in my microbiome data that are easily interpretable by other people. The best solution I’ve come across is simply projecting the data onto a drawing of my organism. Like this:
Alpha diversity of samples from pieces of a seagrass plant projected onto a drawing of that plant.
I’ve used SitePainter to produce these in the past, and in some ways it’s great. I got the figure I wanted and I’ve received a lot of positive feedback about it. The only problem is it has a steep learning curve and is the most dysfunctional GUI I’ve ever come across (generating the figure above took several days of my time, the first time). So when it became necessary to produce over forty more images like it I decided to search for a better way.
My first instinct was that there should be an easy way…