Plasmid copy-number control and better-than-random segregation genes of pSM19035 share a common regulator

Abstract

Transcription initiation of the copy-number control and better-than-random segregation genes of the broad-host-range and low-copy-number plasmid pSM19035 are subjected to repression by the autoregulated pSM19035-encoded omega product in Bacillus subtilis cells. The promoters of the copS (Pcop1 and Pcop2), delta (Pdelta), and omega (Pomega) genes have been mapped. These promoters are embedded in a set of either seven copies of a 7-bp direct repeat or in a block consisting of two 7-bp direct repeats and one 7-bp inverted repeat; the blocks are present either two or three times. The cooperative binding of omega protein to the repeats on the Pcop1, Pcop2, Pdelta, and Pomega promoters represses transcription initiation by a mechanism that does not exclude sigma(A)RNAP from the promoters. These results indicate that omega protein regulates plasmid maintenance by controlling the copy number on the one hand and by regulating the amount of proteins required for better-than-random segregation on the other hand.

Figure 1

Physical map of replication and better-than-random segregation genes of plasmid pDB101. (A) pDB101 has extraordinarily long inverted repeated sequences that comprise about 76% of the plasmid molecule (10). The single-copy DNA is indicated as a broken line. The duplicated sequences are denoted by a continuous line, and by double coordinates. The promoters (Pcop, Prep, PIII, Pδ, Pω, and Pɛ) and the plasmid replication origin (ori) are denoted by filled and empty boxes, respectively. RNA transcripts are indicated by thin arrows, and the gene products (copS, repS, δ, ω, ɛ, and ζ) are denoted by thick arrows. The filled arrows denote previously identified products and the open arrow, the product identified in this work. The SegB region is indicated. (B) Nucleotide sequence of the Pcop (Pcop1-Pcop2), Pδ, and Pω promoters. The transcription start sites of Pcop1, Pcop2, and Pδ and Pω, determined by primer extension assays, are denoted by bent arrows. Conserved −35 and −10 regions are indicated as open boxes. The coordinates are indicated. The heptamers and their relative orientation are indicated by thin arrows below the nucleotide sequence.

Figure 2

Cooperative binding of ω protein to the promoter region of copS (shaded squares), δ (filled circles), and ω (empty rhomboids). DNA fragments spanning the promoter region of copS, δ, and ω genes (0.2 nM) were radiolabeled and incubated with 1 μg of poly [d(I/C)] as nonspecific competitor DNA and an increasing concentration of the ω protein (0.2–200 nM) in buffer C containing 50 mM NaCl for 15 min at 37°C. The signals present in the protein–DNA complex and in the free DNA were determined by densitometry.

Figure 3

Primer extension assay of the Pcop1-Pcop2, Pδ, Pω, and Pαβ in the absence or presence of the ω protein. The supercoiled plasmid DNA (2 nM) was incubated with 20 nM of σ^ARNAP in the absence or presence of increasing concentrations of ω protein (15, 30, 60, and 120 nM) and subjected to in vitro transcription followed by primer extension. In A, Pcop1-Pcop2; in B, Pδ; in C, Pω; and in D, Pαβ as a nonspecific control. The lengths of the cDNAs obtained in the presence or absence of ω are indicated. The length of the standards in the region of interest are indicated. −, absence of protein ω.

Figure 4

The ω protein does not displace σ^ARNAP from the Pcop1-Pcop2 DNA. [α³²P]-Pcop1-Pcop2 DNA (2 nM) incubated with 60 nM ω protein (lane 2) or 60 nM σ^ARNAP (lane 3) or with both the ω (60 nM) and σ^ARNAP (60 nM) (lane 4) and the complexes analyzed by ndPAGE. The free DNA, ω, and σ^ARNAP complexes RNAP (I) and (II) are indicated. + and − indicate the presence or absence of the denoted protein, respectively.

Figure 5

Interaction of σ^ARNAP and the ω protein with Pcop1-Pcop2 DNA. [α³²P]-Pcop1-Pcop2 DNA (2 nM) incubated with 60 nM ω protein (lane 2) or 60 nM σ^ARNAP (lane 3) or with both ω (60 nM) and σ^ARNAP (60 nM) proteins (lane 4). The location of the promoters and heptamers and their orientation and the transcription start sites are indicated at the side of the gel. The position +1 is the transcription start site of the Pcop1 promoter and the +1 position of promoter Pcop2 is denoted between parenthesis. The symbols + and − indicate the presence or absence of the denoted protein, respectively. AG, the A + G reaction loaded in parallel with the samples.