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Gene Expression in Moraxella Osloensis, Photorhabdus Temperata and Xenorhabdus Koppenhoeferi During Host Infection

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Gene Expression in Moraxella Osloensis, Photorhabdus Temperata and Xenorhabdus Koppenhoeferi During Host Infection GENE EXPRESSION IN MORAXELLA OSLOENSIS, PHOTORHABDUS TEMPERATA AND XENORHABDUS KOPPENHOEFERI DURING HOST INFECTION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ruisheng An, M.S. * * * * The Ohio State University 2007 Dissertation Committee: Approved by Dr. Parwinder S. Grewal, Adviser Dr. David L. Denlinger _______________________________ Dr. Saskia A. Hogenhout Adviser Dr. Srinand Sreevatsan Graduate Program in Entomology ABSTRACT Bacterium Moraxella osloensis is associated with slug-parasitic nematode Phasmarhabditis hermaphrodita, and bacteria from genus Photorhabdus and Xenorhabdus are with entomopathogenic nematode Heterorhabditis and Steinernema, respectively. We first determined gene expression of M. osloensis in the mollusk host Deroceras reticulatum by selective capture of transcribed sequences (SCOTS) technique. Two genes, ubiquinone synthetase (ubiS) and acyl-coA synthetase (acs) were up-regulated in both D. reticulatum and stationary phase in vitro cultures. Eleven genes were exclusively expressed in D. reticulatum and were thus infection specific. Mutational analysis on genes of protein-disulfide isomerase (dsbC) and ubiS confirmed that UbiS is important to M. osloensis growth and DsbC serves as a virulence factor. Bacteria Photorhabdus temperata and Xenorhabdus koppenhoeferi, the symbionts of nematodes Heterorhabditis bacteriophora and Steinernema scarabaei, respectively, were virulent to three white grub species Popillia japonica, Rhizotrogus majalis, and Cyclocephala borealis. The median lethal dose at 48h post injection ii and median lethal time at injection dose of 20 cells per grub showed that P. temperata was more virulent than X. koppenhoferi to C. borealis. However, although P. t em p er a t a grew faster than X. koppenhoeferi both in vitro and in vivo, there were no differences in virulence of two bacteria against R. majalis and P. japonica. Using SCOTS technique, 30 genes by P. t e m p er a ta and 25 by X. koppenhoeferi were identified to be differentially expressed during infection to R. majalis. Most genes were unique to either P. t em p er a t a or X. koppenhoeferi, e.g. ttsL encoding a type III secretion system was only identified from P. t empera ta, and lpsE corresponding to lipopolysaccharide synthesis was unique to X. koppenhoeferi. Five nexus genes directly connecting to several other genes were identified as attractive candidates for future studies using the metabolic computational pathway analysis. Compared to the gene expression of M. osloensis, several common genes were found to be up-regulated during infection in all three bacteria, including dsb gene family and genes encoding outer membrane proteins, fatty acid synthesis proteins, and ubiquinone synthesis proteins. iii Dedicated to my parents, wife, and son iv ACKNOWLEDGMENTS I sincerely thank my advisor, Dr. Parwinder S. Grewal, for his great support and guidance throughout my graduate program. His encouragement, enthusiasm, and patience made this dissertation possible. What I have learned from him has prepared me profoundly for future opportunities. I thank Dr. Srinand Sreevatsan for his invaluable guidance and encouragement during my Ph.D. studies. I particularly enjoyed learning the selective capture of transcribed sequences (SCOTS) technique in his lab. I greatly appreciate other members of my advisory committee, Dr. David L. Denlinger and Dr. Saskia A. Hogenhout for their suggestions and encouragement during my Ph.D. studies, and precious comments on my dissertation. I appreciate the valuable help when I walked into their labs to use the equipment. Also I appreciate the helpful comments on the third chapter by Dr. Denlinger, Dr. Michael Sadowsky and Dr. Man-Wah Tan, and the helpful reviews of the fourth chapter by Dr. Erko Stackebrandt. I am grateful to Dr. Ronald B. Hammond for providing useful books on slugs, I am grateful to Ms. Judy A. Smith for help in collecting slugs and to all of my lab colleagues for helping to collect white grubs for my experiments. I am also pleased v by the pleasant working environment in the Entomology Department. I would like to specially express my deepest appreciation to my wife. Without her love and support, I could not go so far. This research was supported by an interdisciplinary grant and a graduate research competitive grant from the Ohio Agricultural Research and Development Center, Wooster, Ohio. vi VITA August 26, 1976..............................Born – Wuzhai, Shanxi Province, P.R. China 1999 ................................................B.S. Plant molecular and developmental biology, Peking University, Beijing, China 2002 ................................................M.S. Molecular biology, Institute of Zoology, Chinese Academy of Scienses, Beijing, China 2002-present ...................................Graduate Research and Teaching Associate, The Ohio State University, Ohio, USA PUBLICATIONS Presentations "Identification of genes transcribed by Moraxella osloensis in slug Deroceras reticulatum using selective capture of transcribed sequences (SCOTS)", 38th SIP, August 7-11, 2005. Anchorage Alaska. "In vivo gene expression of the slug pathogenic bacterium Moraxella osloensis", Wooster Area Molecular Biology Association, October 20, 2006. Wooster, Ohio. "Is it the nematode or the bacteria?" National turfgrass entomologists workshop, Wooster, Ohio, 3/12/2007. HONORS AND AWARDS OARDC Ph.D. graduate research competition grant, 2006 vii Travel award at 2005 International Society of Invertebrate Pathology, 2005 OARDC Annual Conference Graduate Student Research Poster Competition: first place Ph.D. competition, 2005. FIELDS OF STUDY Major Field: Entomology Study in Molecular Microbiology viii TABLE OF CONTENTS Page Abstract........................................................................................................................ii Dedication...................................................................................................................iv Acknowledgments........................................................................................................v Vita.............................................................................................................................vii List of Tables..............................................................................................................xii List of Figures...........................................................................................................xiii Chapters: 1. Introduction............................................................................................................1 References...................................................................................................15 2. A review of the use of selective capture of transcribed sequences technique for studying bacterial infections.................................................................................22 2.1 Summary.....................................................................................................22 2.2 Introduction.................................................................................................23 2.3 Protein based methods................................................................................25 2.4 Mutagenesis based methods........................................................................27 2.5 PCR-hybridization based methods..............................................................29 2.6 SCOTS technique.......................................................................................33 2.7 SCOTS versus other methods.....................................................................39 2.8 Conclusions.................................................................................................40 2.9 References..................................................................................................41 3 Moraxella osloensis gene expression in the primitive mollusk host Deroceras reticulatum............................................................................................................46 3.1 Abstract.......................................................................................................46 3.2 Background.................................................................................................47 ix 3.3 Results.........................................................................................................51 3.4 Discussion...................................................................................................56 3.5 Conclusion..................................................................................................61 3.6 Methods......................................................................................................61 3.7 References...................................................................................................69 4 Three new subspecies of the symbiotic bacterium Photorhabdus temperata: P. temperata subsp. boemarei subsp. nov., P. t em p er a t a subsp. fischerlesauxii, and P. temperata subsp. stackebrandtii subsp. nov.........................................................79 4.1 Abstract.......................................................................................................79 4.2 Introduction.................................................................................................80 4.3 Materials and
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