Successful Establishment of Plasmids R1 and pMV158 in a New Host Requires the Relief of the Transcriptional Repression of Their Essential rep Genes

Abstract

Although differing in size, encoded traits, host range, and replication mechanism, both narrow-host-range theta-type conjugative enterobacterial plasmid R1 and promiscuous rolling-circle-type mobilizable streptococcal plasmid pMV158 encode a transcriptional repressor protein, namely CopB in R1 and CopG in pMV158, involved in replication control. The gene encoding CopB or CopG is cotranscribed with a downstream gene that encodes the replication initiator Rep protein of the corresponding plasmid. However, whereas CopG is an auto-repressor that inhibits transcription of the entire copG-repB operon, CopB is expressed constitutively and represses a second, downstream promoter that directs transcription of repA. As a consequence of the distinct regulatory pathways implied by CopB and CopG, these repressor proteins play a different role in control of plasmid replication during the steady state: while CopB has an auxiliary role by keeping repressed the regulated promoter whenever the plasmid copy number is above a low threshold, CopG plays a primary role by acting coordinately with RNAII. Here, we have studied the role of the regulatory circuit mediated by these transcriptional repressors during the establishment of these two plasmids in a new host cell, and found that excess Cop repressor molecules in the recipient cell result in a severe decrease in the frequency and/or the velocity of appearance of transformant colonies for the cognate plasmid but not for unrelated plasmids. Using the pMV158 replicon as a model system, together with highly sensitive real-time qPCR and inverse PCR methods, we have also analyzed the effect of CopG on the kinetics of repopulation of the plasmid in Streptococcus pneumoniae. We show that, whereas in the absence of CopG pMV158 repopulation occurs mainly during the first 45 min following plasmid transfer, the presence of the transcriptional repressor in the recipient cell severely impairs the replicon repopulation and makes the plasmid replicate at approximately the same rate as the chromosome at any time after transformation, which results in maximal plasmid loss rate in the absence of selection. Overall, these findings indicate that unrepressed activity of the Cop-regulated promoter is crucial for the successful colonization of the recipient bacterial cells by the plasmid.

Keywords: Cop transcriptional repressors; R1 replicon; establishment phase replication; pMV158 replicon; plasmid replication rate; plasmid repopulation.

Figure 1

Schematic comparison of the mechanisms controlling expression of the essential rep gene in R1 and pMV158. In R1, repA is transcribed from P_copB and P_repA promoters, and CopB represses transcription from the stronger P_repA promoter. Gene copA, which encodes an asRNA, overlaps the intergenic region of the copB-tap-repA operon. CopA exerts an indirect control on repA translation by inhibiting synthesis of Tap protein, to which RepA synthesis is coupled. In pMV158, the copG-repB operon is transcribed from the P_cr promoter, which is negatively regulated by transcriptional repressor CopG. asRNAII, transcribed from P_ctII promoter, binds to its complementary sequence in the copG-repB mRNA, thus inhibiting translation of the replication initiator repB gene.

Figure 2

The presence of excess CopB in the recipient cell dramatically and specifically decreases the efficiency of transformation with the R1 replicon. The vertical bar graphs show the number of transformant colonies per ml that appeared after transforming either plasmid-free or pUC18-copB-carrying S. Typhimurium (A) and E. coli (B) cells with DNAs of plasmids pKN1562 (a mini-R1 derivative) or pACYC184 (harboring the R1-unrelated p15A replicon), both of which are compatible with the pUC18 replicon. The same volumes were plated for all transformed cultures; by plating this volume, 500–1,000 p15A-transformant colonies were counted. Transformant colonies were counted after incubation for 24 h at 30°C. The asterisk in (A) indicates the absence of transformants after transforming S. Typhimurium SL1344 carrying pUC18-copB with pKN1562. The ratio (Q) between the number of transformants per ml obtained with pKN1562 and that obtained with pACYC184 is indicated in the graphs on the right of the corresponding vertical bars.

Figure 3

The presence of CopG in the recipient cell decreases the frequency or the velocity of appearance of transformants for the pMV158 replicon in a selective and dosage-dependent manner. (A) The vertical bar graph shows the frequency of transformants after transforming S. pneumoniae harboring different plasmids with pLS1 (pMV158 replicon) or pAMβ1 (pMV158-unrelated replicon). The resident plasmids provided no copG (pC194), inactive copG (pCGA30), and medium and high dosages of active copG gene (pCAG3n and pCGA3, respectively). The ratio (Q) between the frequency of transformants colonies obtained with pLS1 and that obtained with pAMβ1, counted after 60 h of incubation at 37°C, is indicated on the top of the corresponding vertical bars. (B) S. aureus cells, harboring the same set of plasmids as described in (A), were transformed with plasmids containing the replicon of either pMV158 or pT181. Vertical bars represent the frequency of transformants colonies counted after 24 h or 60 h of incubation at 37°C. The down facing blue arrow symbol indicates that the pMV158 replicon is only present as a plasmid cointegrate in the transformants. The ratio (Q) between the frequency of transformants colonies obtained with pLS1 and that obtained with pT181cop608, counted after 60 h of incubation at 37°C, is indicated on the top of the corresponding vertical bars. The asterisk in (B) indicates the absence of transformant colonies appeared within 24 h of incubation. The data presented in this figure summarize the results obtained in typical transformation experiments of S. pneumoniae and S. aureus. Two additional transformation experiments were performed for each species and the results with respect to the inhibitory effect of CopG on the transformation with the pMV158 replicon were similar to those shown here.

Figure 4

Analysis of the plasmid DNA content of the indicated transformant clones grown under selective pressure for both the resident and the incoming plasmid. (A) Pneumococcal cells harboring pCAG3, pC194, pCGA3n, or pCGA30 were transformed with pLS1 and pAMβ1. The gel shows the total DNA content of several transformant clones. Four transformant clones were analyzed for each transformation with pLS1 and only two clones in the case of each transformation with pAMβ1. The combination of recipient strain and incoming plasmid is indicated on the top of the gel. The copG gene content of the resident plasmid in the recipient strain is indicated below the gel image. Homoplasmid strains harboring pCGA3, pC194, pCGA3n, pLS1, or pAMβ1 were used as controls. Supercoiled monomeric forms of the five plasmids are indicated in the gel; monomeric forms of pCGA3, pCGA3n, and pCGA30 have a similar electrophoretic mobility. (B) Staphylococcal cells harboring pCAG3, pCGA3n, pCGA30, or pC194 were transformed with pLS1 and pT181cop608. The gel shows the plasmid DNA content of several transformant clones. Two transformant clones were analyzed for each transformation experiment with pLS1, and one clone resulting from the transformation of S. aureus/pCGA3 with pT181cop608 was also analyzed. The combination of recipient strain and incoming plasmid is indicated on the top of the gel. As in (A), the copG content of the recipient strain in indicated below the gel image. Homoplasmid strains harboring pCGA3 or pLS1 were used as controls. Supercoiled monomeric forms of pLS1 and pT181cop608 are indicated in the gel.

Figure 5

The presence of CopG in the recipient cell unstabilizes the inheritance of the incoming pMV158 replicon in a dosage-dependent manner. The graphs represent the changes in the fraction of transformants retaining the newly-acquired pMV158 replicon (pLS1) when growing for several generations in the absence of selective pressure for the plasmid. The recipient pneumococcal strains lacked copG (A), had a truncated version of copG (B), or harbored a medium (C) or high (D) dosage of the active copG gene. The experimental loss rate (L_ex) of pLS1 was calculated from the slope of the linear regression model of the plot of the experimental values according to Equation (2) (red circles and lines). T₀ and T are, respectively, the fractions of transformants ab initio and after n generations.

Figure 6

The presence of CopG in the recipient cell impairs repopulation of the pMV158 replicon by decreasing the plasmid replication rate. Changes in the fraction of transformants retaining newly-acquired pLS1cop7 (a copy-up pMV158 derivative) were analyzed in pneumococcal strains that either harbor a high dosage of active copG (A) or a truncated version of copG (B). The experimental loss rate (L_ex) of pLS1cop7 was calculated from the slope of the linear regression model of the plot of the experimental values according to Equation (2) (red circles and lines). T₀ and T are, respectively, the fractions of transformants ab initio and after n generations. The graphs in (C,D) show the impact of CopG on the kinetics of pLS1cop7 repopulation. A qPCR approach was used to calculate the variation in the copy number of a specific amplicon of the incoming pLS1cop7 plasmid relative to the chromosome during the growth of the total bacterial population containing pCGA3 (C) or pCGA30 (D) as resident plasmid for 150 min after transformation (left y-axis; blue circle and lines). The pLS1cop7 replication rate (R value), defined as the ratio of plasmid to gDNA duplications, was calculated at different time intervals following transformation of pneumococcal cells harboring pCGA3 (C) or pCGA30 (D). Determination of R was based on the iPCR data of the in vivo plasmid amplification (black circles and lines) and its value (right y-axis) was calculated according to Equation (10). Discontinuous horizontal line in graphs of (C,D) denotes an R value of 1, which characterizes the steady-state plasmid replication. The mean (symbols) and standard deviation (error bars) of all the experimental points in the graphs of (C,D) are displayed. Panels (E,F) show the iPCR analysis of the gDNA samples obtained at the indicated times after transformation of pneumococcal cells carrying pCGA3 and pCGA30, respectively, with pLS1cop7. iPCR assays were carried out by using a pair of divergent primers specific for the pMV158 replicon (Table 3 and G) and the Phusion DNA polymerase. Lane M, DNA molecular weight standard (NZYDNA ladder III; NZYTECH). Note that lanes M are the same in (E,F) because, in fact, both images of these panels arise from the same gel. Dividing lines in (E) indicate grouping of different parts of the same gel. The original image of the gel used for (E,F) composition is shown in Figure S2. A schematic representation of pLS1cop7 displaying the plasmid regions complementary to the divergent primers is shown in (G). Genes copG, repB, and tetL, as well as the dso region, are indicated.

Figure 7

Effect of the velocity of plasmid repopulation on the segregational stability of an incoming plasmid. Two extreme theoretical cases of immediate (A) or null (B) plasmid repopulation are displayed. These models assume that the plasmid does not burden the host cell. We defined “P_n” as the fraction of transformants after n generations; “a” is the initial number of transformants, and “b” is the number of non-transformed cells within the total bacterial population. In the case that immediate repopulation occurs (A), the steady-state plasmid copy number is reached before division of the transformants, and the plasmid is stably inherited to both daughter cells. Dotted circles in (A) represent the number of new plasmid molecules generated in each cell generation. If no repopulation occurs (B), the single, unreplicated plasmid copy is inherited by only one of the two daughter cells resulting from division of the transformants, giving rise to maximal segregational instability.