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  • American Society for Microbiology  (2)
  • Manias, Dawn A.  (2)
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  • American Society for Microbiology  (2)
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  • 1
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 192, No. 6 ( 2010-03-15), p. 1634-1642
    Abstract: The mating response of Enterococcus faecalis cells carrying the conjugative plasmid pCF10 is controlled by multiple regulatory circuits. Initiation of transcription of the prgQ conjugation operon is controlled by the peptide receptor protein PrgX; binding of the pheromone peptide cCF10 to PrgX abolishes PrgX repression, while binding of the inhibitor peptide iCF10 enhances repression. The results of molecular analysis of prgQ transcripts and genetic studies suggested that the elongation of prgQ transcripts past a putative terminator (IRS1) may be controlled by the interaction of nascent prgQ mRNAs with a small antisense RNA (Anti-Q) encoded within prgQ . Direct evidence for interaction of these RNAs, as well as the resulting effects on readthrough of prgQ transcription, has been limited. Here we report the results of experiments that (i) determine the inherent termination properties of prgQ transcripts in the absence of Anti-Q; (ii) determine the direct effects of the interaction of Anti-Q with nascent prgQ transcripts in the absence of complicating effects of the PrgX protein; and (iii) begin to dissect the structural components involved in these interactions. The results confirm the existence of alternative terminating and antiterminating forms of nascent prgQ transcripts in vivo and demonstrate that the interaction of Anti-Q with these transcripts leads to termination via inhibition of antiterminator formation. In vitro transcription assays support the major results of the in vivo studies. The data support a model for Anti-Q function suggested from recent studies of these RNAs and their interactions in vitro (S. Shokeen, C. M. Johnson, T. J. Greenfield, D. A. Manias, G. M. Dunny, and K. E. Weaver, submitted for publication).
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
    detail.hit.zdb_id: 1481988-0
    SSG: 12
    Location Call Number Limitation Availability
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  • 2
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 199, No. 12 ( 2017-06-15)
    Abstract: Tools for regulated gene expression in Enterococcus faecalis are extremely limited. In this report, we describe the construction of an expression vector for E. faecalis , designated pCIE, utilizing the P Q pheromone-responsive promoter of plasmid pCF10. We demonstrate that this promoter is tightly repressed, responds to nanogram quantities of the peptide pheromone, and has a large dynamic range. To demonstrate its utility, the promoter was used to control expression of the toxic peptides of two par family toxin-antitoxin (TA) loci present in E. faecalis , par pAD1 of the pAD1 plasmid and par EF0409 located on the E. faecalis chromosome. The results demonstrated differences in the modes of regulation of toxin expression and in the effects of toxins of these two related systems. We anticipate that this vector will be useful for further investigation of par TA system function as well as the regulated expression of other genes in E. faecalis . IMPORTANCE E. faecalis is an important nosocomial pathogen and a model organism for examination of the genetics and physiology of Gram-positive cocci. While numerous genetic tools have been generated for the manipulation of this organism, vectors for the regulated expression of cloned genes remain limited by high background expression and the use of inducers with undesirable effects on the cell. Here we demonstrate that the P Q pheromone-responsive promoter is repressed tightly enough to allow cloning of TA system toxins and evaluate their effects at very low induction levels. This tool will allow us to more fully examine TA system function in E. faecalis and to further elucidate its potential roles in cell physiology.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2017
    detail.hit.zdb_id: 1481988-0
    SSG: 12
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
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