Francisella tularensis is associated with water and waterways and infects many species of animals, insects, and protists. The mechanism Francisella utilizes to persist in the environment and in tick vectors is currently unknown. We have demonstrated for the first time that Francisella novicida, a model organism of F. tularensis, forms a biofilm in vitro. Selected F. novicida transposon mutants were tested for their ability to form biofilm compared to the wildtype F. novicida strain. Mutation of the putative qseB gene led to an impairment in the ability to form biofilm with no impairment in bacterial growth. A qseC mutant had impaired growth but demonstrated a marked impairment in biofilm production. Mutation in capC affected both bacterial growth and biofilm formation, but no biofilm production impairment was seen with capB or pilE mutants. A deletion mutant in the orphan response regulator FTN_1465, which we propose is the putative QseB, formed significantly less biofilm than the wildtype. When FTN_1465 was complemented back into the deletion mutant, biofilm formation was restored. Thus, the orphan response regulator FTN_1465 is an important factor in biofilm production in vitro in F. novicida. These results demonstrate that Francisella species are able to form biofilms in vitro, suggesting that biofilm formation may be important for the lifecycle of this organism.

The bacterium Francisella philomiragia has been isolated from environmental samples originating from around the globe. F. philomiragia-related strains cause francisellosis of both farmed and wild fish. In addition, occasional human infections caused by F. philomiragia are found in victims of near-drowning and patients with chronic granulomatous disease. We have shown that F. philomiragia forms in vitro biofilms with increased formation at 25 °C over 37 °C conditions. We found that F. philomiragia can form a biofilm in a co-culture with live Acanthamoeba castellanii, an aquatic amoeba. Interestingly, amoeba-conditioned supernatant has an inhibitory effect on production of biofilm by F. philomiragia, whereas Francisella-conditioned supernatant has no effect on growth of amoebae. We have shown that F. philomiragia can infect A. castellanii after only 5 days of co-incubation and that it infects A. castellanii more quickly than the related species F. novicida does. Our studies point to a potentially overlooked interaction between F. philomiragia and Acanthamoeba. This relationship in the marine lifecycle of F. philomiragia may support the persistence of the bacterium in waterways and its ability to infect fish. An understanding of the persistence of this organism in aquatic systems through biofilm formation and its interaction with Acanthamoeba will be important in developing prevention strategies for this pathogen.

Background:The mechanism of Francisella LVS entry into A549 cells is unknown. Results: Microarrays were performed at early infection time points, supported by phenotypic observations and inhibition experiments. Conclusion: Francisella LVS may enter A549 cells by macropinocytosis and quiets the host infection response. Significance: Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell.

We previously showed the feasibility of using locked nucleic acid (LNA) for flow cytometric-fluorescence in situ hybridization (LNA flow-FISH) detection of a target cellular mRNA. Here we demonstrate how the method can be used to monitor viral RNA in infected cells. We compared the results of the LNA flow-FISH with other methods of quantifying virus replication, including the use of an enhanced green fluorescent protein (EGFP) viral construct and quantitative reverse-transcription polymerase chain reaction. We found that an LNA probe complementary to Sindbis virus RNA is able to track the increase in viral RNA over time in early infection. In addition, this method is comparable to the EGFP construct in sensitivity, with both peaking around 3 h and at the same level of infected cells. Finally, we observed that the LNA flow-FISH method responds to the decrease in levels of viral RNA caused by antiviral medication. This technique represents a straightforward way to monitor viral infection in cells and is easily applicable to any virus.

With the increasing popularity of active teaching methods, universities have become more interested in changing instruction from direct lecture to interactive engagement. The George Mason University (Mason, Virginia, USA) Biology Department is achieving this goal through: 1) faculty participation in the SIMPLE project; 2) faculty and student involvement in Discipline-Based Education Research projects; 3) participating in the Learning Assistant program for undergraduate students as scholars; and 4) the Accelerator Program. These approaches are discussed in this paper. Also, interactive teaching strategies, which the biology faculty and graduate teaching assistants use at Mason, are documented in the form of design memos; these memos are introduced in this paper.