CALIFORNIA STATE UNIVERSITY, NORTHRIDGE COMPARATIVE ANALYSIS OF 16S RIBOSOMAL RNA TYPING AND PHYSIOLOGICAL TRAITS WITHIN SPOROSARCINA UREAE A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science in Biology by Tania Kurbessoian December 2016 The graduate project of Tania Kurbessoian is approved: _____________________________________ _______________________ Gilberto Flores, Ph.D. Date _____________________________________ _______________________ Kerry Cooper, Ph.D. Date _____________________________________ _______________________ Larry Baresi, D.P.H, Chair Date California State University, Northridge ii ACKNOWLEDGEMENTS It is with a heavy heart that I write my farewells. To the man in charge and my academic father, Dr. Larry Baresi: It has been three and a half wonderful years since you agreed to have me set foot in your lab; yet it feels like I have known you forever. You have provided me with the skills I can incorporate in the lab as well as in my life. Thank you for sharing your infectious laughter, your inspiring stories, your delicious wine and goodies, handkerchiefs when things got tough, and most importantly-your time. All of which has made my stay at CSUN most excellent. I hope I can make you proud with what I aspire to accomplish! To Dr. Kerry Cooper: You may be new to CSUN, but you are exactly what they needed. Your patience and guidance really helped develop my bioinformatics skills; skills I am hoping to further incorporate into my future and will always be grateful for possessing. Thank you for your time and patience. I can’t wait to hear about the crazy things you do with the Sporosarcina! To Dr. Gilberto Flores: You’re also new at CSUN and I am so happy I had the opportunity to take your seminar class. I never took ecology as seriously as I should have, but your class helped me open my eyes. Thank you for grabbing hold of the reins at the last minute and guiding this project through. I am eternally grateful for your guidance and patience. To Dr. David Bermudes: You helped cultivate my love for mycology and gave me the opportunity to appreciate them- for which I am forever grateful. Thank you. iii To all the other CSUN instructors and professors: Thank you for making my stay at CSUN incredibly memorable. You each have unique talents, quirks and specialties, and I will always miss you all. To the CSUN students: Karen Koch, Ranelle Buck, David Quintero, Alina Adamian, Benham Goldbani, Nicole Fuentes Jaime Lee, Andrew Oliver, Nicholas Rhoades, and Ulises Lopez. You all picked me up and supported me when things got rough. I can’t wait to hear what you all accomplish. To the Planetary Protection officer, Wayne W. Schubert: You were incredibly accommodating and I am so grateful that you took a chance on me and allowed me to work with you the past summer. Thank you for allowing me to use the Bruker MALDI- TOF MS instrument, target and matrix on the Sporosarcina. Many thanks also goes to Heidi Aronson, who was also a pivotal person and taught me how to maneuver the multi- faceted program involved with the Bruker system. And finally to my family: Many thanks, hugs and kisses to my ever patient mother who would sit and endure my many monologues about microorganisms and science. You fueled that drive in me to learn and succeed and I know that nobody could take that away from me. To my brother, your patience and willingness to assist helped make this thesis possible, you have always been my backbone. And finally to HG’s effortless guidance, without whose help it can truly be said this work would have been considerably more difficult. iv Table of Contents Signature page…………………………………………………………………………….ii Acknowledgements………………………………………………………………...…….iii Abstract………………………………………………………………………….....……..vi Introduction………………………………………………………………………………..1 Materials and Methods…………………………………………………………………...16 Results……………………………………………………………………………………21 Discussion……………………………………………………………………………..…31 Conclusion…………………………………………………………………………..…...42 References………………………………………………………………………….….…43 Appendix A: Growth Rates of Sporosarcina…………………………………………….50 Appendix B: Biolog Results…………………………………………...………….….….53 Appendix C: PCA Plots of MALDI-TOF MS Results ………………………………….67 Appendix D: CRISPR/Phage Regions………………...…...…………………..………...68 Appendix E: Plasmid Analysis…….....……………………………………………….....85 v Abstract COMPARATIVE ANALYSIS OF 16S RIBOSOMAL RNA TYPING AND PHYSIOLOGICAL TRAITS WITHIN SPOROSARCINA UREAE By Tania Kurbessoian Master of Science in Biology Sporosarcina ureae is a motile spore forming cocci that traditionally forms packets. The term sarcina is used to describe packets of cocci microorganisms that divide bilaterally, on two or three successive planes. Both physiological and partial 16S ribosomal RNA typing of the ATCC type strain places Sporosarcina ureae in the Bacillaceae family. A key characteristic of the family Bacillaceae is the rod shape and bacterial spore formation. Sporosarcina ureae nomenclature has changed as the result of morphological and biochemical observations. Thus a question arises as to which is a better predictor or tool for classification, physiology (Biolog®), 16S rRNA gene structure, DNA genome sequencing, or MALDI-TOF MS analysis. High throughput Pac Bio sequencing and Biolog® was completed on 6 of the 50+ strains from the California State University, Northridge (CSUN) collection based on extremes of urease activity. MALDI-TOF testing was completed on the entire collection while 16S rRNA sequencing was completed on 33 strains. Based on MALDI-TOF MS work, there are at least 9 different genera, 17 species and 6 strains of Sporosarcina present among the 59 while the 16S ribosomal sequencing found five clusters. Biolog® results created two clusters and vi reaffirmed nutritional work of Pregerson and Risen suggesting Sporosarcina utilizes the Hexose Monophosphate Pathway, Citric Acid Cycle, and Glyoxylate pathways. This study strongly supports the hypothesis that the 16S rRNA gene and physiological studies show a low degree of homology across geographically distributed isolates. The strongest agreement that we could find was between the PacBio 16S consensus sequence and MALDI-TOF MS for P33 and P37. This study also suggests a relatively ancient divergence between Sporosarcina (sarcina) and Bacillus (rod). vii Introduction Sporosarcina ureae (S. ureae) is a gram positive, peritrichously flagellated, spore forming, aerobic cocci, that forms pairs, tetrads or packets of eight (Pregerson, 1973. M.S. thesis, CSUN). CSUN has one of the largest collection of Sporosarcina with 50+ isolates from all over the world in addition to previously isolated ATCC type strains (origins from Martinus Beijerinck and C.B. van Niel). HISTORY Beijerinck was the first to isolate and name the organism Planosarcina ureae from soil samples. In 1903, he proposed that Planosarcina ureae was closely related to other spore forming microorganisms in the genus Bacillus (Beijerinck, 1903). In 1911, Lohnis proposed to name the organism Sarcina ureae due to the sarcina cluster formation. In 1960 researchers MacDonald & MacDonald as well as Kocur and Martinec moved the microorganism to the genus Sporosarcina (first proposed by Orla-Jensen, 1909 and used by Kluyver and van Neil, 1936). Currently in Bergey’s manual, S. ureae rests in the division Firmicutes, the class Bacilli, the order Bacillales, the family Planococcaeceae and the genus Sporosarcina. (Bergey’s, 2009) Bergey’s currently holds nine species in the genus Sporosarcina and as of recently a total of 13 species identified all except for S. ureae are rod shaped microorganisms. The current Sporosarcina species are placed in the genus Sporosarcina mainly due to their spore forming ability and DNA/DNA hybridization techniques comparing these strains with the type strain. The final decision was made mainly due to the spore formation, as well as G+C% content and whole cell fatty acid profiles. 1 Claus determined the composition of S.ureae’s cell walls, contains lysine in the tetrapeptide of the murein with D-glutamylglycine as part of the interpeptide bridge (Claus, 1981). The G+C content is about 40.0-41.5% (Auletta and Kennedy, 1967; Claus, 1981; Bohacek, Kocur and Martinec, 1968; Kocur, Bergan, and Mortensen, 1971) and the endospore is 0.5-1.5µm in diameter (Robinson, 1981; Claus, 1981; Robinson and Spotts, 1983). S.ureae motile by peritrichous flagella. S. urea colonies range from gray or cream and age to a yellow, orange or brown depending on the strain as well as the medium. (Bergey’s 2009) Many different strains of Sporosarcina have been recently isolated, characterized, and added to the genus based on their 16S rRNA gene sequence similarity, DNA-DNA hybridization and spore formation. S. aquimarina is a monotrichoues, gram variable terminal spore forming rod. Colonies are from light orange, smooth, circular to irregular and can grow in the presence of 13% NaCl (Yoon et al., 2001). S. contaminans is a gram positive, strictly aerobic, motile rod with terminal endospores. S. contaminans was isolated from an industrial clean room and formed light beige and circular colonies. (Kampfer et al., 2010). S. globispora originally named as Bacillus globispora is a gram positive or variable peritrichous rod that occurs either singly or in pairs. They form terminal to sub- terminal spores with off-white, irregular lobate colonies. The organism was isolated from soil as well as river water (Larkin and Stokes, 1967; Yoon et al., 2001). 2 S. koreensis is a gram positive terminal spore forming rod that occur singly or in short chains. The colonies are light orange and do not grow in plates more than 7% NaCl and was isolated from Korean soil. (Kwon et al., 2007) S. macmurdoensis is a single, non-motile rod and forms sub-terminal spores. The colonies are white, flat and opaque and the organism can tolerate up to 3% of NaCl. The organism was isolated in the McMurdo Region in Antarctica. (Reddy et al 2003) S. pasteurii initially mentioned as Bacillus pasteurii. The cells are gram positive rods while their colonies are usually circular and glossy. This organism is capable of converting large amounts of urea to ammonium carbonate.