Characterization of Two Aerobic Bacteria from Canada Goose Microflora by Whole Genome Sequence Analyses by Abigail Larkin A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Biology (Honors Associate) Presented July 31, 2020 Commencement June 2021 AN ABSTRACT OF THE THESIS OF Abigail Larkin for the degree of Honors Baccalaureate of Science in Biology presented on July 31, 2020. Title: Characterization of Two Aerobic Bacteria from Canada Goose Microflora by Whole Genome Sequence Analyses. Abstract approved:_____________________________________________________ Patrick Ball The Canada goose (Branta canadensis) monogastric gut is proposed to contain aerobic spore- forming bacteria that produce non-toxin substances that may promote anti-inflammatory immune responses. It is hypothesized that the aerobic bacteria found in samples of Canada goose microflora could have probiotic effects in other avian species or used for commercial or ecosystems analyses. To investigate this, two Gram-variable, spore forming, rod-shaped and chloroform tolerant aerobic bacteria strains A4 and A15 were isolated from fecal samples of resident Canada geese (Branta canadensis). Using a 16S rRNA gene sequences analysis it was determined that both novel strains were 94% similar to other Sporosarcina species rRNA genes and both had their own unique 16S rRNA sequence. Both A4 and A15 were found to contain urease genes, GerA spore germination protein genes, and a fibronectin binding protein. The digital DNA–DNA hybridization (dDDH) of both strains to similar genomes and between each other was <28%, below the 70% cut-off for species definition. In addition, the average nucleotide identity (ANI) and average amino acid identity (AAI) values of both strains were <82% for A4 and <88% for A15, also below the cut-off values for species definition. Phenotypic characteristics were tested, including phenotypic assays testing for 177 different carbon sources and assays testing for pH effects on growth. It was concluded that A4 and A15 represent two novel strains of the Sporosarcina genus, Sporosarcina cascadiensis (A4) and Sporosarcina obsidiansis (A15). Key Words: Microbiome, Spore-forming bacteria, Probiotic, Average Nucleotide Identity Corresponding e-mail address: [email protected] ©Copyright by Abigail Larkin July 31, 2020 Characterization of Two Aerobic Bacteria from Canada Goose Microflora by Whole Genome Sequence Analyses by Abigail Larkin A THESIS submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degree of Honors Baccalaureate of Science in Biology (Honors Associate) Presented July 31, 2020 Commencement June 2021 Honors Baccalaureate of Science in Biology project of Abigail Larkin presented on August 31, 2020. APPROVED: _____________________________________________________________________ Patrick Ball, Mentor, representing the Department of Biology _____________________________________________________________________ Bruce Seal, Committee Member, representing the Department of Biology _____________________________________________________________________ Kristina Smith, Committee Member, representing the Department of Biology _____________________________________________________________________ Toni Doolen, Dean, Oregon State University Honors College I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request. _____________________________________________________________________ Abigail Larkin, Author Acknowledgements Appreciation is extended to Patrick Ball, Ph.D. for taking the time to mentor me throughout this research project and providing feedback throughout the process. Appreciation is also extended to Bruce Seal for his co-mentorship on the project and for taking his time to guide me through this process. In addition, I would like to thank Kristina Smith, Ph.D. for being on the committee and for providing feedback to all of us who participated on the project. I want to thank Brittany Martinez for partnering with me during the research process and for providing funding for the research from the URSA Scholarship. Funding was also provided by my mentor Patrick Ball, Ph.D. though a start-up grant from the OSU Faculty Innovation Committee and the Gaskins Fund. I am grateful for the collaborative effort of all involved and the opportunity I had to work with a group of individuals who helped foster my interest in microbial research. Introduction It was proposed that the Canada goose’s (Branta canadensis) monogastric gut contains aerobic bacteria that produce non-toxin substances that may promote anti- inflammatory immune responses in other avian species. The Canada goose is one of the most common waterfowl species in North America and has a gut flora which contains diverse communities of bacteria [1]. Like many domestic and free ranging bird species the Canada goose’s gut flora plays an important role in host nutrition and protection from pathogens [2]. The purpose of this study was to genotypically and phenotypically characterize two bacterial strains isolated from the Canada goose’s feces. In particular, the study focused on strains of bacteria that exhibit sporulation, a mechanism that enables spore-forming bacteria to survive in diverse environments [3]. The results of the study found two novel bacteria strains, isolated from Canada goose feces, and selected them for their potential to stimulate anti-inflammatory responses in avian species. The novel bacterial species, identified using whole genome sequencing, belong to the genus Sporocarcina and exhibited a unique 16S rRNA sequence with only a 94% similarity to previously identified Sporosarcina species. Sporosarcina characterization and diversity The genus Sporosarcina, which belongs to the family Bacillaceae, was created by Kluyver and van Neil to accommodate bacteria that have spherical or oval-shaped cells, low DNA G+C content (40–42 mol%), and MK-7 as the major menaquinone [4]. Currently Sporosarcina includes 17 species (https://www.bacterio.net/genus/sporosarcina) isolated from a range of environments including some species, such as S. ureae which utilizes the enzyme urease to breakdown urea [5]. Additionally, some species such as S. newyorkensis form endospores. S. newyorkensis is a gram-positive, endospore-forming rods that originated from veterinary clinical specimens in New York State, USA and from raw milk in Flanders, Belgium [6]. Methods of Isolation and Identification Fresh feces from geese were aseptically collected from resident Canada goose populations in Bend, OR USA (44.0582° N, 121.3153° W) utilizing sterile plastic bags. Fecal material was stored in an ultra-cold freezer (- 80 °C) until processed for selection of bacterial spores. Goose fecal material was thawed and suspended in phosphate-buffered saline (PBS) using organic solvent-resistant, polypropylene 15 or 50 ml conical centrifuge tubes followed by vortex mixing for five minutes [7]. Subsequently, low-speed centrifugation was conducted at 1,000 x g for five minutes to eliminate solids. Following centrifugation, chloroform was added to a concentration of approximately 3 percent; e.g., 0.3 ml per 10 ml of fecal suspension or 1.5 ml chloroform per 50 ml fecal suspension and placed on a laboratory shaker for 30 minutes to eliminate vegetative bacterial cells and select for bacterial spores [8 9]. Aliquots of spore suspensions (150µl) were cultured aerobically for two days utilizing brucella agar with blood and vitamin K-hemin (BBHK) or reinforced clostridial agar with L- cysteine, Na acetate, starch without polymyxin B. This procedure resulted in fifteen aerobic bacterial isolates [10]. Two of the reported isolates were propagated on BHI media for preservation. Gram stains performed on both isolates initially indicated they were Gram negative bacilli (Fig 1). DNA was extracted from bacterial colonies using either the QuickExtract™ Bacterial DNA Extraction Kit or the mBio UltraClean® Microbial DNA Isolation Kit. Genomic DNA was used to amplify 16S rRNA genes using the primers 27F (5'AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') [11]. After amplification and analysis of the 16S rRNA, two Gram-variable bacilli, designated A4 and A15, were selected due to the fact that they had the most unique 16S rRNA sequences, that was also 94% similar to previously identified Sporosarcina spp. A4 A15 Fig.1 Gram staining of isolates A4 and A15 showing both strains as Gram negative to Gran Variable Genome Features Genomic DNA was sent to the Bioinformatics Center at the Oregon State University Corvallis campus for whole genome sequencing (WSG) using Illumina’s MiSeq platform 150 bp paired end sequencing chemistry. Raw fastq files were used for de novo genome assembly with St. Petersburg genome assembler (SPAdes) v 3.10.1 [12]. Assembled scaffolds of strains A4 and A15 genome sequences were submitted to National Center for Biotechnology Information (NCBI) for Basic Local Alignment Search Tool (BLAST) analyses using Microbial genomes BLAST analysis tools [13] Phylogenetic analyses of genomic data demonstrated that the isolates were closely related to the genus Sporosarcina, but only 79% related to currently classified Sporosarcina spp. in the database, suggesting the bacteria may be a previously unclassified species (Fig. 2). Three genes