Bailey Benson and Dr. David Erickson, Department of Micro and Molecular Biology

Introduction

Yersinia pestis — the causative agent of the deadly bubonic plague, which killed over one-third of Europe in the 14th century – spreads quickly from person to person due to its ability to create a biofilm, characterized by bacterial adhesion to themselves and surfaces. The bacteria infects fleas and forms a biofilm in their midgut, which causes the flea to both feed more often and cough up bacteria biofim into each new host. In this way, the formation of a strong biofilm is essential for the rapid spread of the bubonic plague. In my experiment, I analyzed which genes would most hinder or help the bacteria Yersinia pestis to create such a biofilm, by studying mutants of the most recent relative of Y. pestis, Y. pseudotuberculosis. The mutants (csrB::Tn5, barA::Tn5, uvrY::Tn5, and rcsB::Tn5) were created through transposon mutagenesis, and the name of each gene (csrB, barA, uvrY, rcsB) corresponds to the gene that was interrupted, thus rending it functionless. By studying these mutants and comparing them to the wild type strain, I can deduce which of these four genes inhibits biofilm production, stmulate biofilm production, or has no effect. Previous research in my lab showed qualitative results (by growth on congo red agar) that suggested the csrB mutant creates a strong biofilm, in comparison to the wild type. The csrB mutant appears dark red, whereas the wt appears almost white, suggesting that the csrB takes up more dye than the wt and thus creates a stronger biofilm. My goal in this project is to quantify those earlier findings.

Methodology

Several different procedures were tried in order measure the amount of biofilm that each strain of bacteria produced. However, all but the last procedure produced inconclusive data. The procedure that yeiled the best results with the smallest standard deviations is as follows. Cultures of wt Yersinia pseudotuberculosis and csrB::tn5 Y. pseudotuberculosis were inoculated in 5 mL of TB broth and shook at 210 rpm for 48 hours at room temperature. 5 uL of each bacteria culture was placed on black filters on plain TB agar and grew for 72 hours. The black filters (which now have bacterial growth on them) were then placed in 50 mL conical tubes (on the side of the tube with the bacteria facing the inside) which contatained 20 mL of PBS (phosphate-buffered soy) liquid. The tubes were taped horizontally to the shaker, so that the bacteria culture face up, and the PBS covered it. The tubes shook at 50 rpm in 5 minutes increments, where the absorbance of the solution was measured at 600 nm every five minutes, in order to measure the amount of biofilm dispersal. Each time the absorbance was measured, the volume that was used to take the measurement was replaced.

Results

The following graph illustrates the results of this experiment:
The y-axis is the absorbance OD₆₀₀

The x-axis is the time at which the absorbance was measured, in minutes since the tubes started to shake (at 50 rpm).

Discussion

As can be seen from the graph, the absorbance of the wt solution is generally higher than that of the csrB solution. This suggests that it was more difficult for the csrB biofilm to disperse, meaning the biofilm created by the csrB is stronger than that of the wt. This finding not only coincides with the qualitative congo-red binding results from before, but also supports our lab’s hypothesis that csrB (a small noncoding RNA molecule) is an inhibitor of biofilm formation. These results support that hypothesis since the csrB mutant, where the csrB gene has been interrupted and thus rendered nonfunctional, shows stronger binding than the wild-type.

Conclusion

I found that the csrb::Tn5 mutant has a stronger biofilm than the wt pseudotuberculosis, as was expected form earlier, qualitative research.