Date of Award

Spring 2006

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Louis Tisa

Abstract

Photorhabdus is a bacterial symbiont of soil nematodes and a lethal pathogen of insects. Many pathogenic or symbiotic bacteria utilize various methods of motility to reach favorable conditions, colonize a host, or have motility genes that also regulate virulence expression. It is not known how motility is regulated, or how it may confer an advantage, in the complex life cycle of Photorhabdus..

We characterized motility in Photorhabdus and found that the bacterium was motile both by swimming (movement in liquid) and swarming (movement on surfaces) under appropriate conditions. Both types of motility utilized the same peritrichous flagella and shared genetic components. However, unlike swimming, swarming behavior was a social form of movement in which the cells coordinately formed intricate channels that covered a surface. The optimal conditions for motility were established including a Na + or K+ requirement. Interestingly, microarray experiments imply that NaCl and KCl regulate motility posttranscriptionally and not at the gene expression level. This ionic salt posttranscriptional regulation of motility has not been observed in other bacteria. We suggest that this form of regulation may be beneficial for an organism that must adapt quickly to changing environments.

To identify the genes involved in motility, P. temperata mutants with altered motility behavior were generated with random transposon mutagenesis. An rssB mutant that displayed a hyperswarming phenotype was isolated, suggesting that RssB acts as a negative regulator of swarming behavior. A yidA mutant, whose function remains unknown, had inhibited swimming behavior and dramatically attenuated virulence. A plu3723 mutant (a luxR homolog) was isolated, that unlike the wild-type, was able to swim without NaCl or KCl. All together 86 motility mutants were isolated and physiologically characterized. Since many of the motility mutants had concomitant changes in expression of antibiotics, hemolysins, proteases, and insect virulence, expression of motility genes may be co-regulated with expression of virulence enzymes in Photorhabdus. The mutants isolated in this study will be useful long-term tools for additional experiments.

The ability of Photorhabdus to swarm could provide a rapid and coordinated colonization of either nematode or insect host, or in traveling from one host to another. The nematode environment is low in nutrients, ionic salts, and amino acids, while the insect hemocoel is high in these solutes. When the nematodes release their bacterial symbionts into the insect hemocoel, the bacteria are exposed to the ionic salts that would induce the flagella regulon. Ecologically, it would be beneficial for the bacteria to be motile upon entering the insect to rapidly colonize the hemocoel. If aspects of virulence expression are co-regulated with motility genes as this research suggests, expression of virulence factors would also be induced upon exposure to the insect. The data presented in this study are the first steps for elucidating a model of motility in the life cycle of this insect pathogen and nematode symbiont.

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