Katie Ballering

E-mail: balle028@umn.edu

Thesis Advisor: Gary Dunny

Year entered: 2004

Degrees received:
B.S., Bacteriology, University of Wisconsin-Madison, 2004

Honors and Awards:

• MPGI travel grant (2006)

• MICaB travel grant (2007)

• ASM General Meeting travel grant (2007)

• Student Representative (2007-8)

• Dennis W. Watson Fellowship (2007-08)

• Student representative to the University of Minnesota Board of Regents Equity, Access and Diversity committee (2006-2010)

• Microbial and Plant Genomic Institute RA Fellowship (2009)

Thesis research:
Enterococcus faecalis is an important vector for dissemination of antibiotic resistance through horizontal gene transfer and is one of the top three nosocomial pathogens in the US. E. faecalis strains are known to form biofilms on biotic and abiotic surfaces and these biofilms may play a role in enterococcal virulence and horizontal gene transfer. To gain an understanding of the genetic mechanisms allowing the formation of a biofilm and the role of biofilm formation in E. faecalis pathogenesis we used a genetic method, Recombinase-Based In Vivo Expression Technology (RIVET), that allows for the identification and characterization of biofilm-specific gene regulatory circuits during all stages of biofilm development. From this screen we identified a putative transcription factor that we named EbrA (enterococcal biofilm regulator). We confirmed the differential expression of ebrA and its importance in biofilm formation in this organism with an in-frame deletion of the ORF and also qRT-PCR. The ebrA null mutant was reduced in biofilm formation by more than 90%. This defect was complemented by expressing the cloned EF_1809 gene in trans from a plasmid. The biofilm expression this transcript was shown to be 12.5 times that of planktonic cells. Based on these results we hypothesize that expression of EbrA is activated following the initiation of biofilm growth, and that it regulates expression of other genetic determinants required for full development of biofilms. The pET28b+ over expression system was used to add a 6-his tag to the N-terminus of the amino acid sequence which allowed us to purify the protein from E. coli. Using the purified protein we were able to examine its DNA binding capabilities with gel shifts using DNA probes that spanned the intergenic region directly preceding the ebrA ORF. These experiments coupled with the results from a GFP reporter construct made using the ebrA intergenic region to drive reporter expression, suggest that EbrA participates in positive auto-regulation. We are currently extending these studies to identify other EbrA targets in the E. faecalis genome. Understanding the mechanism of action of this transcription factor will enhance understanding of enterococcal biofilms..

Publications:

Ballering, K.S., C.J. Kristich, S.M. Grindle, A. Oromendia, D.T. Beattie, and G.M. Dunny. (2009) Functional Genomics of Enterococcus faecalis: Multiple Novel Genetic Determinants for Biofilm Formation in the Core Genome. J. Bacteriol. 191:2806-2814.