Direct evidence for control of the pheromone-inducible prgQ operon of Enterococcus faecalis plasmid pCF10 by a countertranscript-driven attenuation mechanism

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).

FIG. 1.

Scheme for providing Anti-Q in trans to P_Q transcripts and sequence of prgQ and Anti-Q region. (A) E. faecalis cells with pBK1. pBK1, indicated by a circle, contains P_Q and prgQ sequences through IRS1. Nascent prgQ transcripts can either form a terminator (1) at IRS1, causing termination and generating Qs, or an antiterminator (2), which allows transcription of the lacZ reporter (see Fig. S4 in the supplemental material). (B) E. faecalis cells with pBK1 and pDM4. pDM4 contains P_X and Anti-Q sequences and is able to provide Anti-Q in trans (gray transcript, 3), which interacts with nascent prgQ transcripts (4), preventing antiterminator formation and causing termination at IRS1. This leads to reduced transcription of the lacZ reporter. (C) Sequence of pCF10 region used in this study. The sequence of the prgQ sense strand is indicated in normal font, and the sequence of Anti-Q is indicated in bold font. The prgQ and Anti-Q-prgX transcriptional start sites are shown. Anti-Q is derived from the 5′ end of a longer prgX transcript. The prgX ORF, not shown on this map, begins at prgQ −193. Mutations that are used in this study are indicated by arrowheads pointing away from the sequence. Landmarks, such as the 5′ or 3′ end of a plasmid or deletion, are indicated by arrowheads pointing toward the sequence. The positions listed above and below the sequence are relative to the prgQ and Anti-Q transcriptional start sites, respectively. The PrgX binding sites, XBS1 and XBS2, are shown with bars, and the putative antiterminator is shown with two dotted arrows. The putative terminator, IRS1, is shown with two solid arrows. The peptide sequence of the prgQ ORF, which produces iCF10, is indicated.

FIG. 2.

Effects of mutations within prgQ on expression of a downstream reporter. (A) Reporter plasmids and prgQ transcripts with the proposed antiterminating and terminating structures. prgQ and lacZ mRNA are shown in black, and Anti-Q is shown in gray. The location of the poly(U) tract is indicated by a “U,” deletions are indicated by gaps, and point mutations are indicated by arrowheads. pBK1 is the wild-type reporter, pBK1-18 contains a deletion within IRS1 (Δ354-376), pBK1-4 contains mutations within IRS1 (339-41GGG→CCC and 369-71CCC→GGG), and pBK1-21 contains mutations within the antiterminator and IRS1 (197-99CUC→GGG, 339-41GGG→CCC and 369-71CCC→GGG). (B) Levels of transcription past IRS1 as measured by qRT-PCR of lacZ are shown, normalized to the results for a representative pBK1 sample. Strains tested were E. faecalis OG1Sp bearing the indicated plasmid or a plasmid that is identical except for containing the antiterminator deletion (AT deletion [Δ120-253]). Strains were cultured and tested as detailed in Materials and Methods. Error bars indicate one standard deviation from the mean. (C) Diagram of proposed antiterminating and terminating structures for pBK1-1, a reporter identical to pBK1 except for carrying the AT deletion.

FIG. 3.

Effects of the YUNR mutation on Anti-Q-mediated attenuation. Levels of transcription past IRS1 as measured by qRT-PCR of lacZ are shown, normalized to the results for a representative pBK1 sample. Strains tested were E. faecalis OG1Sp bearing the indicated plasmids. pDM4-3 is a derivative of pDM4 with a T:C substitution at the “U” position in the YUNR motif. pBK1-24 is a derivative of pBK1 with a mutation (A215G) that complements the point mutation in pDM4-3. Strains were cultured and tested as detailed in Materials and Methods. Error bars indicate one standard deviation from the mean.

FIG. 4.

Termination and antitermination occur in vitro. (A) Phosphorimager scan of products from a representative IVT assay resolved by gel electrophoresis. Transcripts terminated at IRS1 are indicated (Term), as are runoff (R.O.) transcripts that extend to the end of the DNA template. The frequencies of termination in the reactions shown were 54% with no exogenous RNA, 87% with 150 nM Anti-Q, and 51% with 150 nM human 18S rRNA. (B) The results of three independent IVT assays were analyzed. The average percentages of transcripts that terminate at IRS1 are shown. Error bars indicate one standard deviation from the mean.