WESLEYAN UNIVERSITY Ecological dimensions of significance in diversification of Bacillus subtilis and Bacillus licheniformis Sarah M. Kopac I would first like to thank my thesis committee, Annie Burke, Michael Singer, Danny Krizanc, and especially my adviser, Fred Cohan. They have all shaped my thinking and ability to critically look at the work of others as well as my own, not only through classes but also through their personal mentoring. Fred especially helped me learn how to develop my ideas, has encouraged me to explore my interests in astrobiology and has given me the great gift of acknowledging my management skills, which I would not have touted on my own. Second, I credit my family with always supporting me and encouraging my love of science. It began with studying the Challenger, the Apollo program, and with classic science fiction movies, and has endured to become a meaningful career. My mother has always been there to commiserate with me when I am overwhelmed, my dad has been there for career and travel advice, and my sister has been there to commiserate when mom and dad get annoying. All three of them, throughout these six years, never ceased to remind me that I am special no matter what, and I think they actually believed it. My grandmother has also been an unswaying source of encouragement. She is the grandmother everyone wants to have; always believing that I have been wronged and have never been the perpetrator of a wrong, reminding me to take care of myself, asking if I have enough money to pay the rent, and sending me cards without any occasion. She is simply the best. My friends and former roommates Nikki Evensen and Erica Stankard will always have a very special place in my heart. Swapping complaints about research with Nikki has been my consolation that I am not alone in this. Erica has been my shortest friend for a long time and can i | P a g e always, always make me laugh, nearly never answers the phone except when I am crying or it is 2am, and has never doubted me. My Wesleyan friends, especially Michelle Kraczkowski, have actually made parts of this whole process enjoyable. Kate Miller and Sarah GW are notable for their skills in dancing, and (lack of) trivia knowledge. Jane Wiedenbeck is an inspiration, endlessly energetic, wise, and losing her phone, all of which I love her for. Those who I have worked with in the Cohan lab, especially Melanie Koren, Menherit Goodwyn, Janine Petito and Steffers Aracena, have made science more satisfying. They have all enriched my life and continue to do so. Tod Osier and Jen Klug were my advisers from Fairfield University and are responsible for the majority of the good advice that I’ve received in my life. They’re supportive and wonderful, and I hope I never lose touch with them. Lastly, Craig Breitsprecher has been invaluable in his support and endless patience in the last year. He has expanded my cultural knowledge during a phase when most people become very closed off from the world. He is also utterly ridiculous and makes me laugh, encourages me not to back down, and is the best friend I could ask for. Besides Rosie. ii | P a g e Table of contents Abstract iv Introduction 1 Chapter 1: Ecological dimensions associated with bacterial speciation 19 Chapter 2: Bacillus subtilis-licheniformis growth in high copper and high boron 42 conditions Chapter 3: Genomic differences and their effects on independent and competitive 55 growth Conclusions 64 References 67 Appendix 86 iii | P a g e The bacterial domain includes thousands of known species, and likely orders of magnitude more that have not yet been discovered. Little is known about the causes of diversification in bacteria and the environmental factors associated with recent divergences. Ecotypes are bacterial populations that are theorized to be ecologically distinct, representing the most recent products of speciation. Our study utilizes a unique environmental soil gradient with dimensions such as salinity, boron, and copper decreasing westward along the transect, and other dimensions remaining stable or in random flux along the gradient. We isolated and sequenced a protein coding gene for 620 strains from the Bacillus subtilis-licheniformis clade from the environmental gradient described above. These sequences were used to demarcate ecotypes from the sample set, and tested for associations with the twelve environmental factors measured for the soil samples. We found thirty one ecotypes in our sample set, twenty three in the B. subtilis subclade and nine in the B. licheniformis subclade, including nine previously unidentified ecotypes. The ecotypes are significantly heterogeneous in their associations with iron, F(18, 365)=1.6704, p=0.04239 and boron, F(18, 244)=1.6767 p=0.04401 in the soil, as well as the soil pH F(18, 365)=1.6466, p=0.04699 and the proportion of clay present in the soil F(18, 99)=1.7226 p=0.04753. We further explored the ecological heterogeneity of ecotypes and strains within ecotypes by testing their growth tolerance to high levels of boron or copper in the soil. Ecotypes were marginally significantly different in their growth in high boron media, F(7, 61)=0.0566 p=0.0566, and strains nested within ecotypes were significantly different, F(13, 61) p=0.0087. In high copper media, ecotypes didn’t show differences in growth, F(24, 131) p=0.21975, but strains did, F(73, 82) p=0.00001. This suggests that strains are quickly evolving with respect to their associations with copper and boron, and might gain and lose tolerances within the lifetime of an ecotype. Rapid diversification, even within ecotypes, is supported by a separate project where we compared the genomes content of four strains of Bacillus subtilis subspecies spizizenii within a single ecotype. Gene annotations and genome comparisons with RAST showed that the strains differed in gene content, including genes for carbohydrate usage, specifically in utilization of maltose, maltodextrin and myo-inositol. Only one strain, G1A4, had genes that were non- paralogous to genes in the other strains; these were five genes for maltose and maltodextrin utilization. Strain G1A4 also had two paralogous genes for maltose and maltodextrin utilization in addition to the genes all of the strains shared; the strain G1A3 had three paralogous genes for the utilization of myo-inositol in addition to those in the core genome. To determine if differences in gene content reflect differences in ecology, strains were tested in monocoluture and in competition for their growth in media with the sole energy source as maltose, maltodextrin or myo-inositol. The strain G1A4, predicted to perform best on maltose and maltodextrin, did outperform the other strains. G1A4 also performed better on glucose, indicating that the strain was superior for reasons besides the extra maltose/maltodextrin genes (due to either the five other unique genes with known functions or one of the dozens of unique iv | P a g e genes with unknown functions). The strain G1A3, predicted to perform best on myo-inositol, did not perform the best, even when data was corrected for the strains’ growth differences in the glucose control. These findings demonstrate the value of combining genomic analyses with growth experiments. Differences in the ecology of strains can be difficult to determine from sequence data alone. But taken together, the results indicate that carbohydrates are one factor associated with very recent speciation events in bacteria. v | P a g e Thousands of species of bacteria have been identified in the bacterial domain. These species are divergent in lifestyle and physical structure, including Deinococcus radiodurans, which floats on dust particles and can survive higher levels of radiation than any other known organism (Tempest and Moseley 1982), Aquifex species that tolerate temperatures of up to 95°C (Madigan, Martinko et al. 2005), organisms that can live in single-species ecosystems hundreds of meters under the surface of the earth (Chivian, Brodie et al. 2008), and thioautotrophic organisms that need neither organic carbon nor sunlight to live (Stewart and Cavanaugh 2006). Diversity also exists between more closely related lineages, most notably as ecological diversity within taxonomic species (Welch, Burland et al. 2002, Sikorski and Nevo 2005, Walk, Alm et al. 2007, Walk, Alm et al. 2009, Cohan and Kopac 2011, Luo, Walk et al. 2011). It is accepted that bacterial diversity is associated with environmental factors; light levels, temperature, and phosphorous concentrations in the environment have been linked to divergences in Synechococcus and Prochlorococcus (Allewalt, Bateson et al. 2006, Johnson, Zinser et al. 2006, Martiny, Coleman et al. 2006, Bhaya, Grossman et al. 2007, Martiny, Tai et al. 2009). In this work, we provide evidence that the diversification of Bacillus subtilis and Bacillus licheniformis are associated with environmental dimensions including resource related factors and growth inhibitors. Bacterial diversity is so extensive that its limits have not yet been discovered (Cole, Konstantidindis et al. 2010, Caporaso, Paszkiewicz et al. 2012, Kyrpides, Hugenholtz et al. 2014). As of 2009, there were approximately 1,000 prokaryotic species with sequenced genomes (Wu, Hugenholtz et al. 2009); that number had increased to 1918 (with an additional 989 “permanent draft” genomes) as of 2012 (Pagani, Liolios et al. 2012); 3,285 bacterial or archaeal 1 | P a g e type strains currently have a genome project finished or under way (Kyrpides, Hugenholtz et al. 2014). In addition to named bacterial species, many strains have been identified by environmental DNA and are the only representative in their taxon. For example, in one case, deep sequencing of subsurface soils showed that the most prolific organism is one in a new phylum (Castelle, Hug et al.