Date of Award

August 2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Steven A. Forst

Committee Members

Heather A. Owen, Charles F. Wimpee, Mark J. McBride, Ching-Hong Yang

Keywords

Antibiotics, Bacteriocins, Interspecies Competition, Natural Environment, Phage Tail, Xenorhabdus

Abstract

Xenorhabdus spp. are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. The nematodes penetrate the insect midgut to enter the hemocoel where Xenorhabdus bacteria are released, transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the hemocoel. In addition, different species of nematodes carrying specific strains of Xenorhabdus can invade a single insect. Xenorhabdus spp thereby engage in competition with both related strains and nonrelated gut microbes. In complex media Xenorhabdus spp produce diverse antimicrobial compounds whose functions in biological systems remain poorly understood. R-type bacteriocins are contractile phage-tail-like structures that are bactericidal towards related bacterial species. They are encoded by remnant P2-type prophage in which the C-terminal region of the tail fiber protein determines target-binding specificity. Xenorhabdus nematophila and Xenorhabdus bovienii produce R-type bacteriocins (xenorhabdicins) that are selectively active against different Xenorhabdus and Photorhabdus species. In this study we analyzed the P2-type remnant prophage clusters in the draft sequences of nine strains of Xenorhabdus bovienii. The C-terminal tail fiber region in each of the respective strains was unique, consisting of mosaics of modular units. The intergenic regions between the main tail fiber gene (xbpH) and the sheath gene (xbpS) contained a variable number of genes encoding tail fiber fragment modules that were apparently exchanged between strains. Xenorhabdicins purified from three different X. bovienii strains isolated from the same nematode species displayed distinct activities against each other. Competition experiments revealed that xenorhabdicin activity was predictive of competitive outcomes between two of the strains while other determinants besides xenorhabdicins were primarily involved in the competitive success between the other strains, indicting that several factors may be involved in determining the outcome of competitions between different strains of X. bovienii. Here we show that another species, Xenorhabdus szentirmaii, produced antibiotics that were active against both gut-derived microbes and several species of Xenorhabdus including the well-studied Xenorhabdus nematophila. Antibiotics of X. nematophila were not active against X. szentirmaii. In co-cultures of wild-type X. szentirmaii and X. nematophila in Grace’s medium that mimics insect hemolymph X. nematophila was eliminated. An antibiotic-deficient strain of X. szentirmaii was created by inactivation of the ngrA gene. X. nematophila proliferated in co-cultures with the ngrA strain. In insects co-infected with wild-type X. szentirmaii and X. nematophila the later was eliminated while X. nematophila proliferated in insects co-infected with the ngrA strain indicating that wild-type X. szentirmaii produced sufficient levels of antibiotics to inhibit growth of a related competitor. MALDI-TOF analysis of hemolymph derived from insects infected with wild-type X. szentirmaii revealed the presence of two compounds (m/z 544 and m/z 558) that were absent in hemolymph infected with the ngrA strain. To our knowledge, this is the first study to directly demonstrate that antibiotics determine the outcome of interspecies competition in a natural host environment.

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