A sequence that affects the copy number and stability of pSW200 and ColE1

Abstract

Pantoea stewartii SW2 contains 13 plasmids. One of these plasmids, pSW200, has a replicon that resembles that of ColE1. This study demonstrates that pSW200 contains a 9-bp UP element, 5'-AAGATCTTC, which is located immediately upstream of the -35 box in the RNAII promoter. A transcriptional fusion study reveals that substituting this 9-bp sequence reduces the activity of the RNAII promoter by 78%. The same mutation also reduced the number of plasmid copies from 13 to 5, as well as the plasmid stability. When a similar sequence in a ColE1 derivative, pYCW301, is mutated, the copy number of the plasmid also declines from 34 to 16 per cell. Additionally, inserting this 9-bp sequence stabilizes an unstable pSW100 derivative, pSW142K, which also contains a replicon resembling that of ColE1, indicating the importance of this sequence in maintaining the stability of the plasmid. In conclusion, the 9-bp sequence upstream of the -35 box in the RNAII promoter is required for the efficient synthesis of RNAII and maintenance of the stability of the plasmids in the ColE1 family.

FIG. 1.

Identifying the region in pSW200 that affects plasmid stability. (A) Map of pSW201, pSW203, pSW240, and pSW249. An empty triangle represents a kanamycin resistance gene. (B) Sequence from nt 151 to 400 in pSW200. “−35,” −35 box of RNAIIp; sps, sequence for plasmid stability. (C) E. coli HB101, which contained pSW201, pSW203, pSW240, or pSW249, was cultured and replica plated to determine the numbers of colonies that did not contain a plasmid. The experiment was performed three times. Error bars represent standard deviations.

FIG. 2.

Region upstream of −35 box in RNAIIp and stability of the pSW200 replicon. (A) The region upstream of the −35 box in RNAIIp, from nt 334 to 388, in pSW240 was deleted to generate plasmids pSW241 to pSW250. sps, sequence for plasmid stability (gray box). (B) E. coli HB101, which contained one of these plasmids, was cultured and replica plated to determine the numbers of colonies that did not contain a plasmid. The experiment was performed three times. Error bars represent standard deviations.

FIG. 3.

Sequence essential to the stability of pSW200. (A) The sps region in pSW245 was mutated by nucleotide substitution or insertion. “−35” represents the −35 box in RNAIIp. sps, sequence for plasmid stability. (B and C) Stability of plasmids was evaluated in E. coli HB101. Cells were cultured and replica plated to determine the numbers of colonies that did not contain a plasmid. The experiment was performed three times. Error bars represent standard deviations.

FIG. 4.

Copy numbers of pSW240M and pYCW301M. E. coli HB101(pACYC184) was transformed with pSW240 (lane 1) or pSW240M (lane 2) (A) or pYCW301 (lane 1) or pYCW301M (lane 2) (B). Plasmids were isolated from the cell and cut using SmaI (A) or SacII (B). DNA was then separated by agarose gel electrophoresis. The intensity of the plasmid bands was determined using Gel-Pro software.

FIG. 5.

Stabilization of pSW100 replicon by sps. (A) Sequence upstream of −35 region in pSW100K, pSW142K, and pSW142KWR. (B) E. coli HB101(pSW100K), E. coli HB101(pSW142K), and E. coli HB101(pSW142KWR) were cultured and replica plated to determine the numbers of colonies that did not contain a plasmid. The experiment was performed three times. Error bars represent standard deviations.

FIG. 6.

Binding of αCTD to sps. His-αCTD (A) or the N-terminal tag region (Trx-His-S tag) (B), which was purified from E. coli BL21(pWCTD) or E. coli BL21[pET-32a(+)], respectively, was mixed with a probe, p-sps, which contained sps (lane 2) or a mutant probe, p-msps (lane 3), which did not contain sps. Proteins that were bound to the probe were captured using streptavidin magnetic beads and analyzed by immunoblotting using anti-His antibody. Lane 1 was loaded with 250 ng of purified protein.