Browsing by Subject "pCF10"

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    Biochemical Analysis Of Modulation Of Sex Pheromone Production By Prgy, A Structural Homolog Of A Metalloprotease (Tiki) That Modulates Wnt Signaling In Eukaryotes
    (2017-05) Le, Thinh
    Enterococcus faecalis contains mobile genetic elements that can rapidly spread antibiotic resistance and virulence genes through its population by conjugation. The chromosomally encoded pheromone cCF10 (LVTLVFV) induces conjugative transfer of E. faecalis plasmid pCF10. Pheromone inducible plasmids have evolved a highly specific and sensitive response to pheromone to allow their host (donor) cell to sense recipients while minimizing expression of the conjugation genes in the absence of recipients. The pCF10 PrgY protein reduces production of endogenous pheromone by donor cells to prevent self-induction. Recent data suggested that PrgY shares significant homology to the eukaryotic metalloprotease TIKI that has been shown to cleave the amino terminus of mature Wnt proteins, thereby regulates the Wnt signaling. Comparative modeling of PrgY active site revealed that PrgY and TIKI share key conserved residues in the metal-binding catalytic core as well as overall secondary structure. Based on the structural similarity between PrgY and TIKI, we hypothesized that PrgY reduces endogenous pheromone production in donor cells by specifically binding and degrading cCF10 as it is secreted across the cytoplasmic membrane. To test the working model, affinity chromatography and Surface Plasmon Resonance were used to demonstrate the direct interaction between PrgY and cCF10, and their binding affinities. The results of this work revealed that PrgY directly interacts with cCF10. Strong binding between cCF10 and PrgY was observed, and the binding can be saturated at a level comparable to the calculated theoretical maximum assuming 1:1 binding. Mass spectrometry was used to identify possible degradation products of cCF10 in the culture supernatant from a strain expressing PrgY. Peptide LVTL was uniquely identified in the donor culture supernatant expressing PrgY. This suggests that PrgY cleaved cCF10 after the second leucine, and released the degraded peptide fragments LVTL and VFV that do not have any pheromone activity. The cumulative results of this research provide important insights into the molecular mechanism of PrgY, and advance our understanding on the function of each of the PrgY family members found in a diverse range of species.
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    Reciprocal regulation between the prgQ and prgX operons of pCF10, a conjugative plasmid of Enterococcus faecalis.
    (2011-07) Johnson, Christopher Mark
    Regulation of conjugation of pCF10, a pheromone-response plasmid of Enterococcus faecalis, is a well characterized process that serves as a model for the control of gene expression in bacteria by intercellular signaling. The genes encoded in the pCF10 prgQ operon mediate conjugative transfer of the plasmid in response to a peptide pheromone secreted by plasmid-free E. faecalis cells. The products of the prgX operon, the small RNA Anti-Q and PrgX, the transcriptional repressor of the prgQ promoter, negatively regulate expression of the prgQ operon. Transcription of prgX is initiated at a promoter within the prgQ operon, but oriented in the opposite direction, making transcription of the two operons overlapping and convergent. Each operon encodes a small RNA within its 5' terminus, which is complementary to 5' sequences of the opposing operon. The orientation of the operons and RNA species derived from the 5' terminus of each operon suggested that, in addition to regulation of the prgQ operon by Anti-Q, there may be unrecognized regulatory interactions between the two operons. This led me to undertake a focused study of potential transcriptional and posttranscriptional regulatory interactions between the prgQ and prgX operons. I found that the operons encode mechanisms to negatively regulate each other. Both use a small RNA to reciprocally regulate the expression of downstream genes from the other operon. Notably, each RNA acts via a different mechanism; Qs, derived from the 5' terminus of the prgQ operon, directs posttranscriptional processing of the prgX mRNA by the host factor RNase III. Anti-Q, an RNA derived from the 5' terminus of the prgX operon, negatively regulates transcription elongation of the prgQ operon without the assistance of host-encoded proteins. Additionally, I gained understanding of cis-acting mechanisms that regulate expression of the prgX operon. Specifically, transcription from the prgQ promoter represses the activity of the prgX promoter via a mechanism termed transcriptional interference, and Anti-Q sequences act as an intrinsic terminator, attenuating the expression of prgX mRNA. In addition to elucidating some of the complexity of regulation between the prgQ and prgX operons, this work revealed the high degree of functional efficiency in the RNA sequences of both Anti-Q and Qs. Anti-Q directs its own genesis by acting as an unusual factor-independent termination signal to RNA polymerase and the mature RNA regulates gene expression from the prgQ operon. Qs, for its part, fills at least three roles in the regulation of conjugation, acting as a leader sequence that can attenuate downstream gene expression, a mRNA for the prgQ gene, and a regulatory RNA that directs endonucleolytic processing of its target. Despite the fact that the overlapping sequences between prgQ and prgQ are shared by both operons, the sequences necessary for Anti-Q and Qs to mediate each of their activities are distinct. This raises the possibility that these regulatory functions evolved separately and that the combination of two ancestral regulatory pathways gave rise to the pheromone-response circuit of pCF10. This work has a number of broader implications. It deepens our understanding of how bacteria use RNAs to regulate gene expression. Additionally, the mobile genetic elements of E. faecalis contribute to the evolution of multi-drug resistant E. faecalis and other pathogens. Understanding the molecular mechanisms used by conjugative plasmids may allow the development of novel interventions to prevent this evolution or target bacteria carrying these plasmids.