CALIFORNIA STATE UNIVERSITY, NORTHRIDGE Comparative Genomics and Epigenomics of Sporosarcina ureae A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science in Biology By Andrew Oliver August 2016 The thesis of Andrew Oliver is approved by: _________________________________________ ____________ Sean Murray, Ph.D. Date _________________________________________ ____________ Gilberto Flores, Ph.D. Date _________________________________________ ____________ Kerry Cooper, Ph.D., Chair Date California State University, Northridge ii Acknowledgments First and foremost, a special thanks to my advisor, Dr. Kerry Cooper, for his advice and, above all, his patience. If I can be half the scientist you are someday, I would be thrilled. I would like to also thank everyone in the Cooper lab, especially my colleagues Courtney Sams and Tabitha Bayangnos. It was a privilege to work along side you. More thanks to my committee members, Dr. Gilberto Flores and Dr. Sean Murray. Dr. Flores, you were instrumental in guiding me to ask the right questions regarding bacterial taxonomy. Dr. Murray, your contributions to my graduate studies would make this section run on for pages. I thank you for taking me under your wing from the beginning. Acknowledgement and thanks to the Baresi lab, especially Dr. Larry Baresi and Tania Kurbessoian for their partnership in this research. Also to Bernardine Pregerson for all the work that lays at the foundation of this study. This research would not be what it is without the help of my childhood friend, Matthew Kay. You wrote programs, taught me coding languages, and challenged me to go digging for answers to very difficult questions. I wish we could always collaborate. To Dr. Melanie Oaks and the sequencing core at the University of California Irvine: thank you for the help in generating all this data and getting this project off the ground. Lastly, to my loving parents, Dan and Evelyn, and brother, Kevin. I am so lucky to call you family. iii Dedication This thesis is dedicated to my parents, Dan and Evelyn. I put years of work into this, but it pales in comparison to the work you put into raising me. I love you both. iv Table of Contents Signature page ii Acknowledgments iii Dedication iv List of Figures/Tables vi Abstract vii Comparative Genomics and Epigenetics of Sporosarcina ureae 1 Introduction 1 Methods 7 Results 14 Discussion 18 Tables and Figures 25 References Cited 39 Appendix 1: Supplemental Tables and Figures 48 v List of Figures/Tables Figure 1: 16S rRNA tree of family Planocococcae 25 Table 1: General genome characteristics of Sporosarcina ureae 26 Figure 2: COG graph of Sporosarcina ureae 27 Figure 3A: 16S rRNA tree of genus Sporosarcina 28 Figure 3B: Core genome tree of genus Sporosarcina ureae 29 Figure 4: ACT plot of six strains of Sporosarcina ureae 30 Figure 5: Identity heatmap of species Sporosarcina ureae 31 Figure 6: Core genome tree of Sporosarcina ureae 32 Figure 7: Circos plot of Sporosarcina ureae S204 33 Figure 8: Epigenome map of Sporosarcina ureae 34 Figure 9: Spore heatmap of Sporosarcina ureae 35 Figure 10: Urease loci alignment 36 Figure 11A: MUSCLE alignment of mreB gene 37 Figure 11B: MUSLCE alignement of rodA gene 38 Figure S1: MATLAB model for core 48 Table S1: Methylation data on the six strains of Sporosarcina ureae 49 Table S2: COG groups of the six strains of Sporosarcina ureae 50 Figure S2: Python script for COG generation 51 vi Abstract Comparative Genomics and Epigenetics of Sporosarcina ureae By Andrew Oliver Master of Science in Biology Sporosarcina ureae is an aerobic, motile, spore-forming Gram-positive cocci that was originally isolated in the early 20th century from soil enrichments with elevated levels of urea. The species is unique in that it is the only known spore-forming cocci, and is currently placed in a genus exclusively composed of bacilli. Current research has been focused on the biotech potential of the unique outer cell surface layer (S-layer), and the ability to efficiently convert urea into ammonia. Specifically, researchers are using organisms that hydrolyze urea in applications such as self-healing concrete, biofuel production, and more efficient means to make fertilizer. The goal of this study is to utilize Pacific Biosciences (PacBio) DNA sequencing technology to generate complete genome sequences and to investigate genetic and epigenetic variations between strains of S. ureae that differ in their spatial and temporal isolation. We have sequenced the first six vii complete genomes and methylomes of S. ureae. Genomes were assembled using PacBio SMRT Analysis (v2.3.0) and Geneious (Biomatters; v9.0.4) software programs, and annotated using the Prokaryote Genome Automatic Annotation Pipeline . The average S. ureae genome is 3.3 Mb in size, and contains an average 3160 CDS, 66 tRNAs and 8 rRNAs, while only one of the strains contains a plasmid (64 kb). Epigenetic analysis, using SMRT Analysis and REBASE (New England Biolabs), of the strains demonstrated evidence of several novel adenine and cytosine methylases present in S. ureae. Examination of the species requirement of 97% sequence identity across the 16S rRNA gene was met by all six strains. However, further analysis using in silico DNA-DNA hybridization (DDH), average nucleotide identity (ANI), and additional core- and pan- genome analysis demonstrated a highly divergent species or possibly some of the strains were a subspecies or new species. Further genetic analysis of the entire genus is needed to determine exactly how S. ureae, a spore-forming cocci, relates to the other spore- forming bacillus species in the genus Sporosarcina. Utilizing genomics, our analysis has begun to clarify the make up of the genus, and also found that there may be additional species of spore-forming cocci other than just S. ureae. viii Comparative Genomics and Epigenomics of Sporosarcina ureae Introduction Sporosarcina ureae is an aerobic, motile, spore-forming Gram-positive coccus that was originally isolated in the early 20th century from soil enrichments with elevated levels of urea. Although discovered more than a century ago, its phylogenetic placement compared to other closely related species remains contentious. Martinus Beijerinck originally isolated a motile coccus that clustered in packets and had the ability to form endospores that he named Planosarcina ureae (Beijerinck 1901). In 1911, Lohnis suggested a name change to Sarcina ureae (Pijper, Crocker et al. 1955), while Orla- Jensen in 1909 and Kluyver and van Niel in 1936 both proposed Sporosarcina ureae (Orla-Jensen 1909) (Kluyver and van Niel 1936). In 1963, Kocur and Martinec concluded that a new genus Sporosarcina should be created in the family Bacillaceae, and that genus contain the species S. ureae (Kocur and Martinec 1963). Further research into various molecular and biochemical properties supported the move into the family Bacillacae (Kluckhohn 1986), however further phylogenetic analysis has moved S. ureae into its current family Planococcaceae. Exactly how Sporosarcina ureae fits into the genus Sporosarcina is still somewhat of an enigma. According to the most recent edition of Bergey’s Manual of Systematic Bacteriology, the genus Sporosarcina is composed of nine species, eight of which are bacilli (Vos, Garrity et al. 2011). Indeed, S. ureae is currently the only member of the genus Sporosarcina that is a coccus and organizes itself in a sarcina grouping. All members of the genus form endospores, are catalase positive, motile and all but one species has the ability to hydrolyze urea, although Bergey’s Manual suggests that the 1 strongest evidence that links these organisms together is the similarity in the16S rRNA gene (Vos, Garrity et al. 2011). The rod morphology is a well-studied phenotype. Despite morphology being a trait that necessitates many proteins working together, rodA and mreB are two of the most well studied proteins implicated in the rod-shape morphology (Henriques, Glaser et al. 1998, Daniel and Errington 2003). rodA in B. subtilis and the rodA-pbp2 operon in E. coli are partially responsible for the rod shape in those organisms and when under expressed or knocked out, the cells become spherical (Henriques, Glaser et al. 1998). Another gene, the actin homologue mreB, is necessary for the rod-shape in Caulobacter crescentus (Figge, Divakaruni et al. 2004). Other proteins such as mreC and mreD are also thought to be involved in cellular morphology; however, their exact functions remain unknown (Errington 2015). Additionally, it has been postulated that mbl, along with another protein, mreBH, has some functional similarity to mreB, though more research is needed to elucidate their exact mechanisms (Abhayawardhane and Stewart 1995, Kawai, Asai et al. 2009). Analysis of genes involved in cell shape in S. ureae or other potential cocci shaped Sporosarcina sp. may yield clues as to why they are phylogeneticaly grouped with rod-shaped bacteria. S. ureae is currently the only known spore-forming cocci, and sporulates in harsh conditions and can remain viable for up to a year (Claus, Fritze et al. 2006). Currently, we have very limited data on the sporulation process of S. ureae, although it appears that the sporulation process in S. ureae closely resembles that of bacilli organisms in the genus Bacillus. However, there is one exception or major difference in the process, which is how the cell differentiates into a spore: Bacillus differentiates asymmetrically while S. 2 ureae differentiates symmetrically (Zhang, Higgins et al. 1997). Further work is needed to truly understand how cells of different morphology differentiate into endospores. Another key characteristic of S. ureae is the ability to produce at least one urease, an enzyme that converts urea to ammonia, which studies have shown has higher activity compared to other purified bacterial ureases (McCoy, Cetin et al. 1992). Despite similarities in urease production and sporulation, different strains that were designated by using Bergey’s Manual as S.