Enterococcus faecalis is a Gram-positive bacterium and a member of the normal flora of the intestinal tract. It is also capable of causing opportunistic infection and has gained notoriety recently because of a propensity to rapidly accumulate antibiotic resistance genes. These genes are commonly located on mobile genetic elements that facilitate their dissemination to other bacterial species. The objective of this proposal is to determine the molecular mechanism of action of a genetic locus, par, required for the stable inheritance of the pheromone-responsive conjugative plasmid pAD1. Par operates via a post-segregationat killing mechanism, programming for death any cell that loses the plasmid. Par is regulated by a unique mechanism involving inhibition of toxin translation by interaction of its message with an antisense RNA at multiple dispersed sites and the formation of a stable complex. The goal of Aim 1 of this proposal is to determine the mechanism by which the complex is activated for translation in plasmid-free segregants. This will involve transcriptional analysis of the par RNAs, identification of sequence elements and RNases involved in complex degradation, and evaluation of sequence elements required for stability of the toxin mRNA. Expression of the par toxin, Fst, disrupts the integrity of the host cell membrane. The goal of Aim 2 of this proposal is to determine the mechanism of action of Fst. Two models of Fst function, one involving processing by cellular proteases and one involving selective action at the internal surface of the cytoplasmic membrane, will be tesed. In addition, the characteristics of Fst-resistant mutants will be determined in an effort to identify the specific target of Fst. Finally, saturation mutagenesis will be performed on Fst in an effort to identify which amino acids are essential for its activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055544-07
Application #
7086259
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Rhoades, Marcus M
Project Start
1999-05-01
Project End
2008-06-30
Budget Start
2006-07-01
Budget End
2008-06-30
Support Year
7
Fiscal Year
2006
Total Cost
$228,531
Indirect Cost
Name
University of South Dakota
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
929930808
City
Vermillion
State
SD
Country
United States
Zip Code
57069
Weaver, Keith E (2015) The Type I toxin-antitoxin par locus from Enterococcus faecalis plasmid pAD1: RNA regulation by both cis- and trans-acting elements. Plasmid 78:65-70
Brinkman, Cassandra L; Bumgarner, Roger; Kittichotirat, Weerayuth et al. (2013) Characterization of the effects of an rpoC mutation that confers resistance to the Fst peptide toxin-antitoxin system toxin. J Bacteriol 195:156-66
Weaver, Keith E (2012) The par toxin-antitoxin system from Enterococcus faecalis plasmid pAD1 and its chromosomal homologs. RNA Biol 9:1498-503
Johnson, Christopher M; Manias, Dawn A; Haemig, Heather A H et al. (2010) Direct evidence for control of the pheromone-inducible prgQ operon of Enterococcus faecalis plasmid pCF10 by a countertranscript-driven attenuation mechanism. J Bacteriol 192:1634-42
Shokeen, Sonia; Johnson, Christopher M; Greenfield, Tony J et al. (2010) Structural analysis of the Anti-Q-Qs interaction: RNA-mediated regulation of E. faecalis plasmid pCF10 conjugation. Plasmid 64:26-35
Weaver, Keith E; Kwong, Stephen M; Firth, Neville et al. (2009) The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids. Plasmid 61:94-109
Shokeen, Sonia; Greenfield, Tony J; Ehli, Erik A et al. (2009) An intramolecular upstream helix ensures the stability of a toxin-encoding RNA in Enterococcus faecalis. J Bacteriol 191:1528-36
Weaver, Keith E; Reddy, Shirisha G; Brinkman, Cassandra L et al. (2009) Identification and characterization of a family of toxin-antitoxin systems related to the Enterococcus faecalis plasmid pAD1 par addiction module. Microbiology 155:2930-40
Shokeen, Sonia; Patel, Smita; Greenfield, Tony J et al. (2008) Translational regulation by an intramolecular stem-loop is required for intermolecular RNA regulation of the par addiction module. J Bacteriol 190:6076-83
Francia, Maria Victoria; Weaver, Keith E; Goicoechea, Patricia et al. (2007) Characterization of an active partition system for the Enterococcus faecalis pheromone-responding plasmid pAD1. J Bacteriol 189:8546-55

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