Cells were labeled with fluorescently tagged wheat germ agglutinin (WGA), which binds the bacterial cell envelope; multiple complementary gating strategies were employed for robustness

Cells were labeled with fluorescently tagged wheat germ agglutinin (WGA), which binds the bacterial cell envelope; multiple complementary gating strategies were employed for robustness. Here, we sought to (i) simultaneously assess the presence of RNAs produced from the proximal (early induced transcripts [early Q]) and distal (late Q) portions of the operon in individual cells, (ii) investigate the prevalence of heterogeneity in induced transcript length, and (iii) evaluate the temporality of induced transcript expression. Using fluorescent hybridization chain reaction (HCR) transcript labeling and single-cell microscopic analysis, we observed that most cells expressing early transcripts (QL, operon does not account for failure OSI-906 of induced donor cell gene transfer. is definitely enabled by manifestation of plasmid genes encoding adherence, type IV secretion, and DNA control machinery (Fig. 1A). Manifestation and transfer are induced when recipient cells transmission donors via the cCF10 (C) peptide (1). Induced manifestation happens from promoter PQ, but basal manifestation also occurs from this promoter in the absence of induction by C (Fig. 1B). Without C, transcripts from PQ terminate in the 1st inverted repeat sequence (IRS1) and produce QS transcripts (2). Upon induction by C, the rate of recurrence of transcription from PQ raises, and transcripts continue past IRS1 and through the entire conjugation operon (3). Here, we considered the possibility that some induced cells are not be able to functionally conjugate due to premature induced transcript termination, resulting in induced transcripts of varied lengths (Fig. 1B). In this work, the term QL will be used to refer to induced transcripts terminated at IRS2, and QOp (Q operon) will be used to refer to all transcripts extending recent IRS2. Furthermore, early QOp will be used to discuss downstream genes encoded proximal to IRS2, while late QOp will be used to refer to genes encoded distally (15 to 30 kbp downstream). Promoter PQ manifestation is regulated from the pCF10-encoded PrgX protein (Fig. 1B). The activity of PrgX is definitely modulated from the cCF10 (C) and iCF10 (I) peptide pheromones. PrgX complexed with C allows induced transcription from PQ, while PrgX complexed with results in improved promoter repression (4). I is definitely produced from pCF10 via translation of mRNA originating from PQ and generally serves to limit manifestation of the conjugation machinery in the absence of potential plasmid recipients and to shut down the response following induction. Open in a separate windows FIG 1 Maps of pCF10 and the pCF10 regulatory region. Transcripts demonstrated are indicated from promoter PQ, with or without induction by C. (A) Map showing the prolonged QOp transcript and genes for which HCR transcript-labeling probes were designed. Early QOp transcripts (QL transcripts are generated from the opposite strand and lengthen from promoter Px, terminating near the Defb1 3 end of (green lollipop). and upstream loci in the OSI-906 5 section of the operon. Additionally, we have not yet examined multiple induced transcripts within the same cells. The published data are consistent with models where induced manifestation of a full-length QOp transcript could create the necessary conjugation machinery from a single initiation event. However, the rate of recurrence of transfer from highly induced OSI-906 donor populations is generally below 10?1 (6) (further examined below); this suggests the possibility that only a portion of the population induced for manifestation of early QL transcripts actually expresses mRNA from the entire operon. The lack of thorough characterization of downstream gene manifestation motivated us to further investigate manifestation and conjugation ability at both the populace and single-cell levels. In this work, OSI-906 we sought to investigate whether (i) all cells induced at promoter PQ are capable of expressing the full QOp transcript or (ii) whether some cells communicate only partial QOp transcripts terminating before the 3 end of the operon (Fig. 1B). Note that in the second scenario, we would expect to find substantial numbers of induced donors not expressing 3 transcripts. This subpopulation of cells could.