B-264. Assessing the Role of the Hfq Protein in Francisella tularensis

J. R. Chambers, K. S. Bender;
Southern Illinois Univ., Carbondale, IL.

Francisella tularensis is the causative agent of the disease tularemia and a potential biological weapon with the capability of becoming fatal following the inhalation of only ten or more bacteria. This ‘Category A’ biothreat agent is known to inhabit both a wide range of environments and hosts, yet most of its basic regulatory pathways have yet to be elucidated. In many other pathogenic bacteria, the global regulator Hfq has been shown to play a vital role in survival and virulence. Hfq has also been shown to interact with small non-coding RNAs (sRNAs), an alternative regulatory phenomenon which has recently been shown to play an important role in a myriad of cellular activities. To better understand alternative regulatory mechanisms of F. tularensis subsp. novicida strain Utah 112, phenotypic and gene expression analyses were performed on two different hfq mutants possessing a single transposon insertion (EZ-Tn5 ) in either the 5´ (hfq2) or 3´ (hfq1) region of the hfq gene (Gallagher, et al). Growth rates in rich tryptic soy (TS) medium at 37°C exhibited a marked decrease for the hfq2 mutant compared to that of the wild type (U112) and hfq1 mutant. A similar disparity was found when growth rates were monitored in TS medium under acidic conditions (pH of 5.6). When grown at 42°C, the difference between U112 and hfq2 rates became even more pronounced and a slightly slower growth profile for hfq1 was found. Thus, the location of transposon insertion into the hfq gene affected the strain to differing degrees. RT-PCR results from corresponding RNA extractions indicated a differential expression pattern for hfq in U112 when grown at either pH 5.6 or 42°C. Expression of mglA, a transcriptional regulator involved in intramacrophage growth and recently reported to control Hfq in F. novicida, also exhibited a differential expression profile in the mutants compared to U112 under normal laboratory conditions (pH 7.0 and 37°C). Together, these phenotypic and expression data indicate that Hfq and potentially small RNAs play an important role in the growth rate and stress response of Francisella.