Figure 1.
Linear map of plasmids used in this study.
(A) Plasmid pSW200 (4367 bp) has an RNAI-RNAII region, oriV, bom, mobCABD and a region that contains 41 copies of 15-bp direct repeats (DR). (B) Plasmid pSW201 includes the entire pSW200 sequence and a Km-resistance gene (open triangle) that is inserted into the mob region at a PstI site. Plasmid pSW201N is identical to pSW201 except for the inverted DR region. Plasmids pSW210, pSW219, pSW230, pSW231, pSW232 and pSW252 are deletion derivatives of pSW201. Plasmid pSW207 contains 40 of the 41 repeats with the SspI-DraI fragment replaced by a Tc-resistance gene (filled square). Numbers represent the nucleotide positions from the DraI site in pSW200. The map shows the following restriction enzyme sites; A, AflIII; B, BglII; D, DraI; H, HincII; N, NcoI; Ac, AccI; Nh, NheI; P, PstI; S, SspI, and Sp, SpeI.
Table 1.
Plasmids used in this study.
Table 2.
Exclusion of pSW207 by pSW201 derivatives.
Figure 2.
Correlation between number of repeats and competition among plasmids.
Derivatives of pSW201 that contain various numbers of 15-bp repeats were cotransformed with pSW207 into E. coli HB101. The transformants were plated on LB agar and then replica-plated on LB agar that contained Km or Tc to select those that contained pSW201 derivatives with various numbers of repeats (empty column) and pSW207 (filled column).
Figure 3.
RT-qPCR analysis of activity of RNAIp and RNAIIp.
(A) Map of pSW261, pSW262, pSW263, and pSW264. Arrow indicates direction of transcription. Numbers represent relative nucleotide positions in pSW200. (B)(C) RNA that was transcribed from a region in Tc-resistance gene was amplified by RT-qPCR. 16S rRNA was used as an internal control and amounts of mRNA that were transcribed from RNAIp and RNAIIp were normalized to amount of 16S rRNA. Amount of tet mRNA from pSW261 and pSW263 was set to 100%. Experiment was performed three times and each sample was prepared in duplicate. Empty square: a fragment from tetracycline-resistance gene; error bar: standard deviation.
Figure 4.
Transcription from DR and RNAIIp.
Transcriptional fusion was generated by inserting a fragment from nt 1 to 426, which contains RNAIIp in pSW201 (pSW242), and a fragment from nt 3356 to 4367 in pSW201, which contains DR (pSW243), into a luciferase reporter plasmid, pKK175-6-lux. Luciferase activity was monitored with a luminometer and presented in relative light units (RLU). Each experiment was performed three times and each sample in the experiment was prepared in duplicate.
Figure 5.
−10 and −35 sequences in RNAIp, RNAIIp, and DR.
(A) Sequences of -10 and -35 boxes in RNAIp and RNAIIp. (B) Forty-one copies of 15-bp repeats in DR region, from nt 3341 to nt 3955 in pSW200. Sequences that are homologous to the -35 box are underlined.
Figure 6.
Analysis of proteins that bind to 15-bp repeats.
An E. coli MG1655 lysate was mixed with a biotin-labeled probe, DR-I, which contained the entire DR region (lanes 3, 6), or probe 167 (lanes 2, 5). Proteins in the cell lysate that was bound to the probes were captured using streptavidin-coated magnetic beads and analyzed by immunoblotting using antibodies against β subunit of RNA polymerase (RNAP β)) (lanes 1–3) and σ70 (lanes 4–6). Lanes 1 and 4 were loaded with 0.05% cell lysate.
Figure 7.
Binding of RNA polymerase to 15-bp repeats.
(A) Sequences of DNA probes used in EMSA. DR probe contains four copies of 15-bp repeats from nt 3896 to nt 3955. Sequences highlighted in DR probe resemble −35 sequences. Underlined region in DR probe from nt 3900 to nt 3930 was inserted into 167 sequence to yield 167-DR. (B) Purified RNA polymerase holoenzyme (Epicentre) was added to a reaction mixture that contained biotinylated DR probe (lanes 1–7). Unlabeled DR probe was added to compete for binding (lanes 5–7). Probe 167 and 167-DR (lane 8–11) were used to confirm binding of RNA polymerase to repeat region. Protein-DNA complex was separated using a 7% polyacrylamide gel and detected using a LightShift chemiluminescence EMSA kit (Pierce).
Figure 8.
Exchange of RNA polymerase from DR region to RNAIp and RNAIIp.
(A) Sequences of RNAIp and RNAIIp probes. (B) Purified RNA polymerase (RP) was added to 32P-labeled-DR probe (Fig. 7A). Unlabeled RNAIIp and RNAIp probes, which contained sequences shown in (A), were used to analyze exchange of RNA polymerase from DR region to RNAIp and RNAIIp. (C) DNA-protein complexes that contained a biotinylated-DR probe (Bio-DR) and RNA polymerase (Bio-DR/RP) were captured using streptavidin-coated magnetic beads. After unbound RNA polymerase had been removed, 32P-labeled RNAIIp and RNAIp probes were added to RNA polymerase-DR complex to analyze exchange of RNA polymerase from repeats to RNAIp and RNAIIp. Probe 167 was used as a negative control.
Figure 9.
Competition of ColE1-like plasmids by pSW200 repeats.
(A) Three plasmids in the ColE1 family that contains DR – pBR322-210, pACYC-210, and pSW100-210 were tested to determine their capacity to destabilize an incompatible plasmid. E. coli HB101 was transformed with pUC18 (lane 1), pBR322-210 (lane 2), pACYC184 (lane 4), pACYC-210 (lane 5), pSW106 (lane 7), and pSW100-210 (lane 8). The cells were also cotransformed with pUC18 and pBR322-210 (lane 3), pACYC184 and pACYC-210 (lane 6), and pSW106 and pSW100-210 (lane 9). (B) Plasmids pRK-210, a plasmid that contains an RK2 replicon and DR, and pACYC-210 were tested to determine their ability to destabilize a compatible plasmid. E. coli HB101 was cotransformed with pRK-210 and pSW207 (lane 1), pACYC-210 and pSW207 (lane 2), and pRK-210 and pACYC184 (lane 3). Plasmids were isolated using an alkaline lysis method and detected by agarose gel electrophoresis. Asterisks indicate pUC18, pACYC184, and pSW106.