Hello again! Quarter 1 is now officially over, and Superlab wrapped up last Thursday and Friday with poster presentations. Last time I posted, I was on my way home for fall break and didn’t have sequence data yet, so a lot has happened since then. Three weeks ago, we sent 11 different samples off for sequencing, which came from 6 different original bacterial colonies. We ended up getting usable sequence data back for all 6 colonies, which was exciting, since we’d been having such bad luck up to that point. As it turned out, the six colonies we were able to sequence corresponded to the following bacterial species: sample 3 was Enterobacter cloacae, sample 10 was Sphingomonas aerolata, sample 12 was Pseudomonas brenneri, sample 13 was also Sphingomonas aerolata, sample 15 was also Pseudomonas brenneri, and sample 16 was Stenotrophomonas maltophilia (see phylogenetic tree at the bottom for species diversity). As it turned out, these six colonies corresponded to four different species of bacteria. We went on to analyze and compare sequence data from the two other groups in our larger group. One group (KUOUAK samples on the tree) analyzed data from sassafras, a type of dicot, and the other group (DDJJ) analyzed data from a conifer. Unfortunately, none of the sequences from the second bamboo sample came back positive, so we were unable to compare diversity between the two bamboo samples. Of note, many of the samples from both KUOUAK and DDJJ were phylogenetically similar to our bamboo species, indicating that there was a lot of overlap between species among the different types of plants.
The whole point of this quarter of superlab was to analyze the phyllospheric diversity of plants on Haverford’s campus. Our original hypothesis was that plants in similar macroenvironments, ie, plants that were located close to each other in similar areas hosted similar bacterial populations. Although we have too few samples in our data to really confirm or refute our original hypothesis, based on the data we were able to tentatively conclude that macroenvironment does contribute to the diversity of bacteria on different plants. However, outliers, such as the Curtobacterium ammoniigenes found on the conifer, lend support to the idea that bacterial populations are somewhat dependent on host species. Local host environment of the leaves may play some part in determining what kind of bacteria can survive. For example, most of our bacterial species are typically found in harsh environments, indicating that they may be able to live on the nutrient-poor surface of bamboo leaves. The Curtobacterium found on the conifer typically lives in acidic environments, so it may find the surface of a conifer needle more inviting than a bamboo leaf. Of note, according to primary literature, almost all of our bacterial samples are typically found in aqueous environments, and it was really humid and rainy the week we originally collected samples, indicating that the water might have attracted the bacteria, or could have washed them onto neighboring plants. Also of note, almost all of our bacteria were gram-negative bacilli, indicating that gram-positive bacteria may have been resistant to PCR or other techniques used throughout the quarter. In conclusion, while we can neither confirm nor refute our hypothesis, preliminary data lends support to the idea that both macroenvironment and microenvironment of the plant leaves has a role in determining phyllospheric diversity.
If we had another seven weeks, there are a couple of things that we would want to try with this experiment. First of all, we would re-run our culture-independent analysis so that we would be able to sequence a wider-range of bacteria. We would also try different methods to try to get more gram-positive samples, such as boiling samples before PCR at a higher temperature. We would also want to re-run analysis of bamboo leaves from other parts of the plant, which would give us a better idea of whether macro or microenvironment played a larger role in the determination of bacterial diversity. From there, we would want to analyze and compare the phyllospheres of bamboo plants in other locations, both on and off campus. We would also want to compare diversity on different parts of the plant during different seasons and weather conditions.
Overall, this was a great quarter. I came into the year knowing little about bacteria, and hardly anything about plants, and I feel like I really learned a lot about both in the past seven weeks, and I can’t wait to take quarter classes in the spring to learn more! Not only did I learn a lot about the concepts behind what we were doing, but I became much more comfortable with lab techniques pertaining to both, which will definitely be useful in the future. Beyond concepts and lab techniques, I got more experience with analyzing data, making posters and presenting our findings to people both in and outside of the scientific community, which I think is a really important skill to have, and why superlab is such a fantastic course!
The second quarter of superlab was supposed to start today, but due to Hurricane Sandy it’ll start Wednesday. I’m not sure if there’ll be a blog for the quarter or not, so stay tuned, and for now enjoy pictures of our phyllogenetic tree and poster!