Figure 1.
Genetic map of the pLS20cat conjugation region and summary of the transcriptional lacZ fusions used in this study.
A. Map of the conjugation region of plasmid pLS20cat. Positions and directions of the genes and the positions of the predicted transcriptional terminators are indicated with arrows and lollipop symbols, respectively. Panels B and C show a blow-up of the 600 bp rcoLS20 gene 28 intergenic region and the different fragments fused to lacZ. Fusions in (B) and (C) were used to study activities of the promoters Pc and Pr, respectively. Features of the intergenic region are given on the top line. Numbers correspond to the bp position in this region. Names of the fragments cloned are indicated. The small triangles indicate position of an inverted repeated sequence (see text). Strains containing Pc-lacZ fusions in combination with the Pspank-rcoLS20 cassette were grown on plates containing 10 µM or 1 mM IPTG. The symbols “+++”, “+”,and “−” reflect intense blue, pale blue, and white colonies after growth on X-gal containing plates. Colors of the colonies were observed after 16 and 48 hours of incubation at 37°C for strains containing pLS20cat or the Pspank-rcoLS20 cassette, respectively.
Figure 2.
Correlation between the kinetics of Pc promoter activity and conjugation efficiencies of pLS20cat.
Overnight cultures of the strain PKS8 (F_Ic-lacZ, pLS20cat) and recipient strain PS110 were diluted to an OD600 of 0.05. Next, samples taken at different times were used to determine conjugation efficiency of pLS20cat by a standard conjugation protocol (continuous line), and the promoter Pc activity by measuring β-galactosidase activity (broken line). T = 0 corresponds to the end of the exponential growth phase. The presented graph corresponds to a representative experiment. The experiment was carried out three times and the corresponding values differed by less than 10%.
Figure 3.
Promoter Pc is located 461 bp upstream of the start codon of gene 28 and overlaps with the divergently oriented Pr promoter.
A. Determination of promoter Pc and Pr sequences by deletion analysis and primer extension. pLS20cat containing cells harvested at the end of the exponential growth phase were processed to isolate their total RNA, which was used in primer extension assays as described in Materials and Methods. Features of the promoter Pc are shown above the sequence. The dotted vertical lines and black straight lines indicate the 5′ end points of the transcriptional lacZ fusions present in strains GR68 and GR70, displaying and not displaying promoter activity, respectively. The core promoter and the putative upstream UP element is indicated by a light blue box; the −35 and −10 hexamers, and the extended −10 motif are indicated with dark blue and green boxes, respectively. The transcription start site determined by primer extension and the direction of transcription are indicated with the corresponding encircled base and a black bent arrow. The thin grey bent arrow corresponds to the 3′ end point of the smaller extension product that coincides with the start of an inverted repeat which is marked with a pair of thin blue arrows above the sequence. Features of promoter Pr are shown below the sequence. The dotted vertical lines and the black straight lines indicate the 3′ end points of the transcriptional fusions with lacZ reporter present in strains GR82 and GR116, displaying and not displaying promoter activity, respectively. The deduced position of the Pr core promoter, and the −35 and −10 boxes are indicated with orange and red boxes, respectively (see text). The transcription start site determined by primer extension and the direction of transcription are indicated with the corresponding encircled base and a black bent arrow. B. Primer extension to determine the transcription start sites of promoters Pc (left panel) and Pr (right panel). The cDNA products of the primer extension reactions are indicated with bent arrows (lane P). Free lane in which no sample was run is indicated with “−”. Lanes M, M1 and M2 correspond to [G+A] chemical sequencing reactions of a short 230 bp DNA fragment corresponding to the studied pLS20cat region obtained by PCR amplification as described in Materials and Methods. In the case of the Pc promoter, a smaller extension product with a relatively strong signal was observed 37 bp downstream of the extension product shown. The longer extension product most likely reflects the correct transcription start site based on the following arguments. First, it is known that AMV reverse transcriptase prematurely terminates cDNA synthesis when reaching a stem loop in the RNA, and that the prematurely terminated molecules map at the bottom of the secondary structure [71], [72]. The position of the strong signal coincides with the 3′ end of an inverted repeat (indicated in Fig. 3A). Second, no putative core promoter sequences are evident upstream of the 5′ position of the shorter extension product. Third, if the stronger signal corresponds to the transcription start site, the responsible promoter would be present on Fragment VIIIc used for the transcriptional lacZ fusion in strain GR70. However, no promoter activity was observed with this strain (see text). And fourth, the transcription start site based on the longer extension product corroborates the RNAseq data. C. Schematic overview of RNAseq expression data of pLS20cat genes rcoLS20 and 28 under conditions with (top panel) and without overexpression of rcoLS20 (lower panel). The amount of right and leftward “reads”, given in green and red, respectively, are presented on a log2 scale. The positions of the divergently oriented genes rcoLS20 and 28 are indicated on the top with a red and green arrow, respectively. Dotted lines and black arrows indicate the approximate start sites of the divergent transcripts driven by the Pc and Pr promoters.
Table 1.
Pr promoter activity at different induction levels of RcoLS20.
Figure 4.
Analysis of the RcoLS20 binding sites in the rcoLS20 - gene 28 intergenic region by EMSA.
A. Schematic representation of the DNA fragments used as probes and a summary of the EMSA results. Top panel. A map of the 600 bp rcoLS20-gene 28 intergenic region is shown as a black line alongside with the positions of the characteristics in this region. The divergently oriented genes rcoLS20 and 28 are indicated with a red and blue arrow, respectively. The triangles indicate ribosomal binding sites. The position of the Pc and the Pr promoter (boxed) and their transcription start sites (bent arrows) are indicated in blue and red, respectively. The position of the unique EcoRI site in this region is also indicated. Lower panel shows the different fragments used in EMSA; the names of the DNA fragments, the levels of RcoLS20 binding and the numbers of shifts are indicated on the right. Asterisks indicate positions of introduced mutations. B. Examples of the EMSA results obtained using DNA fragments described in panel A. The left-most lane of each panel corresponds to samples loaded without protein. Increasing concentrations of RcoLS20 were prepared using a two-fold dilution method, and ranged from 0.212–6.8 (7 lane panels) or 0.95–7.3 µM (5 lane panels).
Figure 5.
Identification of a conserved motif constituting (part of) the RcoLS20 binding site.
A. Schematic representation of the region corresponding to Fragment V, which encompasses promoters Pr/Pc and the identified repeated motif that form (part of) the binding site for RcoLS20. Top line. Position of promoters Pc and Pr are indicated in blue and red, respectively. The position of the unique EcoRI site is indicated. A 13 bp long direct repeat (5′-TCAGTGAAAAAAA-3′) is indicated with rightward directed red arrows. The leftward-directed arrow indicates the position of the complementary 9 bp sequence 5′-TTTCACTGA-3′. Second line (Fragment V). Arrows indicate the positions of the identified motifs a1–a7 and b1–b4. Motifs present on the upper and the lower strand are shown in green and purple, respectively. Third and fourth line show identified motifs present on Fragment III_A and XII_A, respectively. Black, dark grey, grey and light grey indicate motifs identical to the consensus sequence or deviating at one, two or three positions, respectively. B. An alignment of the nucleotide sequences of the eleven identified motifs and their flanking sequences. Names according to the nomenclature in “A” are given together with information on the strand and the region. Sequences corresponding to the consensus sequence of the motif are given in bold. C. A representation of the consensus motif generated by Weblogo [73]. The size of each nucleotide corresponds to the frequency with which that nucleotide is observed in that position.
Figure 6.
Footprint analyses of the binding of RcoLS20 to the rcoLS20 - -gene 28 intergenic region.
Fragment V, end-labeled at the Pc template strand, was analyzed for binding of RcoLS20-His. First lane (−) was not incubated with protein. Concentrations of RcoLS20-His, increasing by two-folds, ranged from 0.11 to 7.04 µM. The positions of the Pc and Pr promoters are indicated on the left. Bars on the right reflect the regions covered by Fragments IIIA (F_IIIA) and XIIA (F_XIIA). Positions of motifs a1–a7 and b1–b4 are indicated with green or purple arrows at the right.
Figure 7.
The 75 bp spacer region separating operators OI and OII contains a static bent.
A. Schematic representation of the region encompassing operators OI and OII and the regions corresponding to the DNA fragments amplified by PCR that were subjected to 2% agarose (B) and 8% native PAA (C). Position of the Pc/Pr promoters and the RcoLS20 binding motifs within the operators are indicated with grey rectangles and arrows, respectively. The 75 bp region separating OI and OII is shown as an interrupted line and the position of the unique EcoRI site is given. The predicted curvature in this region is represented by the blue arc above the top line, and by a blue shading in the equivalent region in fragments A–C. Fragments A–C were run on 2% agarose or on 8% native PAA gel followed by ethidium bromide staining.
Figure 8.
RcoLS20 forms tetramers in solution.
The oligomerization state of RcoLS20 protein in solution was studied by two complementary analytical ultracentrifugation assays. A. Sedimentation velocity assay. Sedimentation coefficient distribution c(s) profile corresponding to 10 µM purified RcoLS20. B. Sedimentation equilibrium assay. Upper part: Sedimentation equilibrium data for RcoLS20 (empty circles) are presented together with best-fit analysis assuming protein dimer (dashed line), tetramer (black line), or octamer (dotted line) species. The data indicate that RcoLS20 is a tetramer at 10 µM. Lower part: The difference between estimated values and experimental data for protein tetramers (residuals).
Figure 9.
Model of the different layers contributing to the genetic switch controlling expression of the pLS20 conjugation genes.
A. RNA polymerase acts itself as a switch because it is unable to bind simultaneously to both of the two overlapping and divergently oriented promoters. Consequently, RNA polymerase (the brown ellipse shaped form) binds only one promoter at a time resulting in transcription of only the gene(s) controlled by this promoter. B. RcoLS20 generates a self-sustaining positive feedback loop by activating transcription from its own promoter (Pr) (left panel). This, combined with the simultaneous repression of the divergent conjugation promoter (Pc), results in conjugation being maintained effectively in the “OFF” state. Relief of RcoLS20-mediated repression of the Pc promoter results in activation of the conjugation genes (right panel). In addition, this interrupts the auto-stimulation of the Pr promoter, preventing further synthesis of RcoLS20, which in turn will contribute in pushing and maintaining conjugation in the “ON” state. The negative auto-regulatory loop of RcoLS20 that probably functions to keep RcoLS20 within a low concentration range (see text) is not presented. C. DNA looping results in a high local concentration of RcoLS20, increasing specificity and affinity that dampens transcriptional fluctuations between and within individual cells (left panel). This would contribute to tight repression of the Pc promoter, keeping conjugation in the “OFF” state under conditions antithetic to conjugation without compromising the ability to switching rapidly to a high expression state (i.e. “ON”, right panel) of the conjugation genes when appropriate conditions occur. rcoLS20 and gene 28, -the first gene of the conjugation operon-, are indicated with large red and blue arrows, respectively. The same coloring scheme is used for the corresponding promoters (rectangular) and transcripts (thin broken arrows). Activation and repression of transcription are indicated with continuous black lines ending in an arrow and a “T” shape, respectively. The red cylindrical structures, which may reflect one or two RcoLS20 tetramers, represent the RcoLS20 oligomer mediating DNA looping.