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Characterization of a Staphylococcus epidermidis RP62A Toxin-Antitoxin System

by
Rohini Roy
B.S., 2003
Rutgers University

Thesis Advisor: Nancy Woychik, PhD

Graduate Program: Molecular Genetics, Microbiology and Immunology

RWJMS Research Tower
7th floor Conference Room

Friday, May 15, 2009
10:00 am


Abstract

Toxin-antitoxin (TA) systems are genetic adaptations employed by most free-living bacteria and comprise a single operon encoding a toxin gene and an antitoxin gene, the products of which interact to form a stable complex. TA systems have a distinct purpose depending on whether they are plasmid-borne or chromosomally encoded. The role of plasmid encoded TA systems is to stably maintain plasmids within the host during replication by post-segregational killing (PSK), a highly effective strategy that results in the death of plasmid-free segregants. Chromosomally encoded TA systems are believed to assist bacteria in their response to environmental stresses such as nutritional starvation, UV damage and exposure to antibiotics. These stresses induce a state of quasidormancy or arrested growth, which conserves nutrients for the remaining population. In response to external stimuli, the ratio of toxin to antitoxin is triggered to favor the toxin since the antitoxin is then susceptible to degradation by activated cellular proteases. Unlike classical exotoxins such as cholera or anthrax, TA toxins function inside the cell by disrupting DNA replication, transcription or translation to rapidly halt cell growth until the stress has passed. If normal growth conditions resume the cell can recover by producing enough antitoxin to counteract the toxin. However, if the stress persists, prolonged exposure to the toxin without renewed antitoxin production can result in toxin-induced cell death, thereby purging the bacterial population of damaged or weakened cells. TA systems have also been implicated in bacterial persistence after antibiotic treatment and in biofilm formation and are thought to be responsible for the switch from active growth to dormancy, which is characteristic of persister cells and cells within a biofilm.
We have identified a potential TA system in a biofilm forming strain of Staphylococcus epidermidis(RP62A) that bears all the hallmarks of a bona fide chromosomally encoded TA system. Interestingly, the TA system is absent in the non-biofilm forming strain ATCC 12228. The antitoxin phd (prevents host death) and the toxin doc (death on curing) are homologous to the phage P1 plasmid-encoded E. coli phd-doc operon. We have found that as with E. coli Doc, RP62A Doc shuts down cell growth by inhibiting protein synthesis. Nonetheless, RP62A Doc acts through a mechanistically distinct pathway from P1 Doc, thereby highlighting the functional difference between chromosomally encoded and plasmid-based TA systems. RP62A Doc also has a highly conserved nine amino acid sequence that is common to all known Docs. We used an attenuated Doc (harboring a substitution mutation in this region) to demonstrate that it binds single-stranded RNA and causes an accumulation of 50S and 30S ribosomal subunits in the cell. Therefore, we propose that RP62A Doc perturbs translation by binding Shine Dalgarno (SD) sequences of cellular mRNA, thereby blocking recognition of the SD by the ribosome and preventing assembly of the 70S, leading to an accumulation of the constituent ribosomal subunits.


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