Identification and Characterization of the Hfq protein and small RNAs in Francisella novicida

Author

Jacob Richard Chambers, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2011

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bender, Kelly

Abstract

Francisella tularensis is the causative agent of the disease tularemia and a potential bioterrorism agent. Few regulators have been identified in this organism and little is known about its genetic regulatory networks. In this dissertation project, culture-based and molecular methods were used to both determine the role of the RNA chaperone protein Hfq and identify potential novel small RNAs in F. tularensis subsp. novicida strain U112. The Hfq protein is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis, and has been shown in several bacteria to interact with small RNAs as a post-transcriptional regulator of mRNA stability and translation. Molecular methods were employed to determine that hfq is potentially transcribed in an operon with both the immediate up- and downstream genes. Phenotypic analysis of two transposon insertions within the hfq ORF revealed that the N-terminal region of the Hfq protein is more important for stress tolerance than the C-terminal end. Complete deletion of hfq resulted in a variety of growth defects under certain stress conditions such as heat-shock, low pH, and oxidative stress. Gene expression of hfq under several of these conditions changed significantly, further suggesting a role for the protein during stress tolerance. Because Hfq likely functions as a global regulator, the expression of several genes in the hfq mutant strain were compared to wild-type and some were significantly altered in particular growth backgrounds. The hfq mutant also exhibited a delayed entry into stationary phase and increased biofilm formation under certain conditions. Shotgun cloning and high-throughput sequencing were used to generate a list of potential sRNAs, an important class of regulators that had yet to be studied in F. novicida. Three candidates were selected and their expression verified using Northern blot analysis and self-ligating RACE. The sRNA transcript designated CISC-1 appears important for certain aspects of cell growth and is differently expressed under several stress conditions. ISC-2 is a transcript that has a minor effect on cell growth during exponential phase, but is upregulated during stationary phase. The third sRNA, ISC-16, is highly conserved among Francisella species and is potentially important for the biosynthesis of bacterial fatty acids. These sRNAs represent an important group of regulators that, along with the Hfq protein, could be important for controlling global gene expression in Francisella.