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Functional dissection of the HigBA module insight into differences between chromosomally and plasmid encoded TA systems

by
Jennifer Marie Hurley
BS, 2004
Juniata College

Thesis Advisor: Nancy A. Woychik, PhD

Graduate Program: Molecular Genetics, Microbiology & Immunology

RWJMS Research Tower
7th Floor Conference Room

Thursday, July 9, 2009
10:00 am


Abstract

The HigBA toxin-antitoxin system (TA) is a TA module encoded both intra and extra chromosomally in free-living bacteria. Originally discovered on the Rts1 plasmid, the module stabilizes Rts1 within the Proteus vulgaris population. However, putative HigBA modules are also encoded within the chromosome of several pathogens including uropathogenic Escherichia coli, CFT073. While the function of extra chromosomal TA modules is well understood, the role of the intra chromosomally encoded toxins is still unclear. It has been proposed that chromosomally encoded TA systems may play a role in the cellular response to stress because the action of TA toxins induces a state of cell growth arrest. Studying the differences between orthologous modules that are intra and extra chromosomally encoded can lead to a better understanding of the function of chromosomal TA modules.

We determined the precise mechanism of action of the HigB toxins from both the Rts1 plasmid and from the chromosome of E. coli CFT073. Though both proteins were demonstrated to be endoribonucleases, there were distinct mechanistic differences between the HigB orthologues. HigB-Rts1 is a highly toxic protein, which inhibited protein synthesis by 80% within 20 minutes of induction. HigB-Rts1 mediated cleavage of 100% of both in-frame and out-of-frame AAA sequences, which coincides with one of the most frequently used codons in the AT-rich Proteus spp., lysine (AAA). HigB-CFT073, however, displayed a much less toxic phenotype when over-expressed in both E. coli K12 and CFT-073; it only mildly inhibited protein synthesis at 4 hours post induction. Although expression of HigB-CFT073 was not extremely toxic, it did exhibit endoribonuclease activity with mild specificity for A-rich regions. The distinct differences between these two orthologues may distinguish the function of the intra and extra chromosomal TA modules; extra chromosomally encoded modules are designed to kill their host in order to maintain in the population, the plasmid on which they are encoded while intra chromosomally encoded TA module are intended to slow growth, allowing the host cell to evade permanent damage during times of stress.


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