Brigham Young University BYU ScholarsArchive All Theses and Dissertations 2017-12-01 Acetobacter fabarum Genes Influencing Drosophila melanogaster Phenotypes Kylie MaKay White Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation White, Kylie MaKay, "Acetobacter fabarum Genes Influencing Drosophila melanogaster Phenotypes" (2017). All Theses and Dissertations. 6613. https://scholarsarchive.byu.edu/etd/6613 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in All Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Acetobacter fabarum Genes Influencing Drosophila melanogaster Phenotypes Kylie Makay White A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science John M. Chaston, Chair Joel Griffitts Laura Bridgewater Department of Microbiology and Molecular Biology Brigham Young University Copyright © 2017 Kylie Makay White All Rights Reserved ABSTRACT Acetobacter fabarum Genes Influencing Drosophila melanogaster Phenotypes Kylie Makay White Department of Microbiology and Molecular Biology, BYU Master of Science Research in our lab has predicted hundreds of bacterial genes that influence nine different traits in the fruit fly, Drosophila melanogaster. As a practical alternative to creating site-directed mutants for each of the predicted genes, we created an arrayed transposon insertion library using a strain of Acetobacter fabarum DsW_054 isolated from fruit flies. Creation of the Acetobacter fabarum DsW_054 gene knock-out library was done through random transposon insertion, combinatorial mapping and Illumina sequencing. Successful mapping of transposon insertion was achieved for 6418 mutants with hits within 63% of annotated genes within Acetobacter fabarum DsW_054. Insertion sites were verified in 40 mutants through arbitrary PCR and sequencing. To test the utility of the library, genes were selected from MGWAS results on host colonization which show LPS pathway enrichment in the significant gene predictions. Genes upstream of Lipid-A creation show significant differences in host colonization whereas downstream genes show no effect. In addition, genes were selected from MGWAS results on Drosophila starvation resistance which show Methionine/Cysteine synthesis, Cobalamin synthesis, and Biotin synthesis pathway enrichment. Under our experimental conditions we could not verify influence of these pathways on host starvation resistance. However, they do appear to influence host colonization abundance. This transposon insertion mutant library will be useful for ongoing research in our lab as well as any field studying Acetobacter species, such as other insect microbiome and fermentation research. Keywords: Acetobacter, Drosophila, microbiota, microbiome, lipopolysaccharide, metabolic pathways ACKNOWLEDGEMENTS I would first like to thank my advisor, John Chaston, for his guidance and support throughout this educational journey. I would also like to thank my committee members, Joel Griffitts and Laura Bridgewater for their support and guidance. In addition, my husband for supporting me in my goal of achieving a graduate degree. TABLE OF CONTENTS TITLE PAGE ............................................................................................................................................................ i ABSTRACT ............................................................................................................................................................. ii ACKNOWLEDGEMENTS ..................................................................................................................................... iii LIST OF TABLES .................................................................................................................................................. vi LIST OF FIGURES ................................................................................................................................................ vii INTRODUCTION .................................................................................................................................................... 1 Host traits influenced by the gut microbiome ........................................................................................................ 1 Metagenome-Wide Association to predict microbial gene effects on host phenotype ............................................ 2 Drosophila as a model organism for microbiome research .................................................................................... 3 Acetobacter species in the microbiome and other disciplines ................................................................................ 7 Lipopolysaccharide effect on microbe-host interactions ........................................................................................ 8 Arrayed mutant libraries as a genetic resource ...................................................................................................... 9 CHAPTER 1 .......................................................................................................................................................... 11 ABSTRACT....................................................................................................................................................... 11 INTRODUCTION.............................................................................................................................................. 12 MATERIALS AND METHODS ........................................................................................................................ 14 Bacterial and fly growth media and conditions. .............................................................................................. 14 Creation of an arrayed mutant library ............................................................................................................. 15 Combinatorial mapping and sequencing: ........................................................................................................ 16 Data analysis: ................................................................................................................................................. 17 Library validation: ......................................................................................................................................... 17 Metagenome-wide-association study and KEGG pathway analysis (host colonization): ................................. 18 Mutant phenotyping in fruit flies .................................................................................................................... 19 RESULTS .......................................................................................................................................................... 20 Creation of an arrayed and mapped transposon insertion library in A. fabarum DsW_054 .............................. 20 Prediction of essential genes in A. fabarum DsW_054 .................................................................................... 22 Prediction of bacterial pathways that influence bacterial load in D. melanogaster .......................................... 23 Validation of MGWA predictions by mutant analysis..................................................................................... 25 iv DISCUSSION .................................................................................................................................................... 27 CHAPTER 2 .......................................................................................................................................................... 31 INTRODUCTION.............................................................................................................................................. 31 MATERIALS AND METHODS ........................................................................................................................ 31 Bacterial and Fly growth media and conditions. ............................................................................................. 31 KEGG pathway analysis of MGWAS predictions .......................................................................................... 32 Mutant phenotyping in fruit flies .................................................................................................................... 32 RESULTS .......................................................................................................................................................... 33 KEGG pathway analysis of genes predicted to influence Drosophila starvation resistance ............................. 33 Mutant phenotyping in flies: CFU abundance ................................................................................................. 34 DISCUSSION .................................................................................................................................................... 35 SUMMARY ........................................................................................................................................................... 38 BIBLIOGRAPHY .................................................................................................................................................