Coxiella burnetii is one of the most infectious pathogens known (ID50=1-10 bacteria). It is able to infect both humans and livestock; in humans it causes Q fever. Due to its low infection dose as well as its incredible resistance to environmental factors, Coxiella is recognized as a potential bio-terrorism agent (class B select agent) and thus is important to study. Coxiella has a biphasic development cycle; it cycles from a stationary phase, small cell variant (SCV), to a developmentally active, log phase, large cell variant (LCV). In this project, I have been investigating Coxiella’s intervening sequence (IVS) within its rRNA gene. IVS’s are selfish genetic elements that disrupt 23S rRNA genes and have to be excised in order to yield a mature, fragmented 23S rRNA. Previous work with Salmonella showed that fragmentation of 23S rRNA by an IVS correlated with enhanced degradation of its 23S rRNA during stationary phase. I hypothesize that IVS-mediated fragmentation of 23S rRNA enhances its degradation during Coxiella’s transition from log-phase (LCV) to stationary-phase growth (SCV). The 23S rRNA of Coxiella was amplified using polymerase chain reaction (PCR). The PCR product was transcribed in vitro and the IVS sequence was excised using RNase III, a cellular rRNA processing enzyme. The fragmented 23S rRNA was used in an in vitro rRNA degradation assay with RNase A. As a control, intact 23S rRNA was also degraded with RNase A. It is hypothesized that because the fragmented 23S rRNA has four termini as a substrate for RNase A, degradation will occur at an increased rate compared to intact 23S rRNA. The results of this experiment will further clarify the purpose of the highly conserved IVS sequence in the Coxiella genome and its role in the development cycle of the bacterium.

*Note: Jeremy St. Goddard will be joining Alec during this presentation. Jeremy did the computational work that led to the biochemical work of this project.

Rift Valley fever is the result of a dangerous virus that infects both humans and livestock and shows potential for global spread. To this end, we are interested in disrupting the interaction of viral nucleocapsid protein, N, with the viral RNA genome, that is necessary for replication. This knowledge could lead to the development of new antiviral therapies. The first steps require an understanding of how the protein recognizes and binds the RNA. We will achieve this via a unique approach using the predictive power of computational science to select novel RNA sequences, or aptamers, that will be synthesized and analyzed for binding to N.

We first programmed an algorithm to look for common features on known N-binding RNA sequences. The core of the algorithm is formed from thermodynamic structure calculations performed by software called FoldAlign; our program integrated and interpreted FoldAlign. We then screened a randomly generated RNA sequence library in silico against the known N-binding RNA sequences. Furthermore, we built a filter within the algorithm to select the highest scoring sequences, as given to us by thermodynamic similarity. These ‘winning’ sequences were then synthesized in the laboratory and their binding properties determined. The relative binding affinities were then compared to the strength of the computational score to determine whether the algorithm was a good predictor of RNA sequences/structures that were recognized in a biochemical assay.

Information from these binding assays will be used to improve the algorithm by integrating in silico sequences that bind well to N protein into the pool of known N-binding RNA sequences, so that we are continually refining the thermodynamic motif(s) that the algorithm looks for. We hope this experiment leads not only to a better understanding of the virus, but also to antiviral therapies that end the deadly and infectious nature of this disease.

In Life’s nearly four billion year history, organizational transitions have occurred that fundamentally altered the course of evolution. One of these, the evolution of multicellularity, occurred independently in at least two dozen lineages, giving rise to a remarkable variety of complex forms. In the volvocine algae, many transitional structures are retained in extant lineages, ranging from the unicellular, flagellated Chlamydomonas reinhardtii to the extravagant Volvox barberi, which contains up to 50,000 differentiated cells. In addition to its normal unicellular state, C. reinhardtii is also capable of plastically forming amorphous multicellular clusters, called palmella, which may be triggered by the presence of grazing predators. The increased size of palmella relative to single cells offers C. reinhardtii protection from grazing predation, so we tested the hypothesis that the propensity to form palmella would increase due to this selective advantage. We performed experimental evolution by continuously co-culturing 8 replicate populations of Chlamydomonas reinhardtii with the predatory ciliate Paramecium tetrauerlia for ~350 generations. Response to selection is assayed via reaction norms for palmella formation, which capture shifts in phenotypic plasticity arising from predation pressure. Reaction norms are constructed by mixing experimental C. reinhardtii isolates with different concentrations of a cell-free filtrate of the predatory culture, and measuring the frequency of palmella formation at each concentration. Previous genomic comparisons show that gene families involved in the formation of extracellular matrix are expanded in Volvox relative to Chlamydomonas. Because of their expansion in the Volvox lineage, genes in these families are suspected of having a role in the evolution of multicellularity, and are likely candidates for control of palmella formation. We investigate changes in these gene families in the experimental populations, providing a mechanistic view into one possible route by which multicellularity can evolve.