Plasmid copy-number control and better-than-random segregation genes of pSM19035 share a common regulator Ana B. de la Hoz*, Silvia Ayora*, Izabela Sitkiewicz†, Silvia Ferna´ ndez*, Renata Pankiewicz†, Juan C. Alonso*‡, and Piotr Ceglowski† *Department of Microbial Biotechnology, Centro Nacional de Biotecnologı´a,Consejo Superior de Investigaciones Cientı´ficas,28049 Madrid, Spain; and †Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland Communicated by Donald R. Helinski, University of California, San Diego, CA, November 10, 1999 (received for review July 10, 1999) Transcription initiation of the copy-number control and better-than- (RNA III) that induces transcriptional attenuation within the leader random segregation genes of the broad-host-range and low-copy- region of the rep mRNA (11, 15–17). In the absence of Cop, the number plasmid pSM19035 are subjected to repression by the auto- increased (derepressed) transcription of rep mRNA interferes, in regulated pSM19035-encoded ␻ product in Bacillus subtilis cells. The cis, with initiation of transcription of RNA III (convergent tran- promoters of the copS (Pcop1 and Pcop2), ␦ (P␦), and ␻ (P␻) genes scription). Thus, the Cop protein limits the amounts of rep mRNA have been mapped. These promoters are embedded in a set of either either directly (as a repressor) or indirectly, by affecting the intensity seven copies of a 7-bp direct repeat or in a block consisting of two of transcription necessary to produce RNA III. The factor(s) that 7-bp direct repeats and one 7-bp inverted repeat; the blocks are would control the synthesis of Cop has not been yet described. ␻ present either two or three times. The cooperative binding of Two discrete regions, SegA and SegB, are involved in the protein to the repeats on the Pcop1, Pcop2, P␦, and P␻ promoters segregation stability of pSM19035 (13). SegA is required for the represses transcription initiation by a mechanism that does not conversion of oligomeric forms into monomers (class a; ref. 5). The exclude ␴ARNAP from the promoters. These results indicate that ␻ SegB region, which assures better-than-random plasmid pSM19035 protein regulates plasmid maintenance by controlling the copy num- distribution, encompasses the ␦, ␻, ␧, and ␨ genes (refs. 13, 14; this ber on the one hand and by regulating the amount of proteins ␦ required for better-than-random segregation on the other hand. report; Fig. 1A). The protein shares a high degree of homology with the family of ATPases involved in partitioning of diverse ͉ ͉ bacterial plasmids and bacterial chromosomes (class c; refs. 13, 18, plasmid replication Gram-positive bacteria protein–DNA ␧ ␨ interactions ͉ transcriptional repressor 19), whereas the and gene products prevent the appearance of plasmid-free segregants (class b; see above) (unpublished results). In this report, we show that a pSM19035-borne gene ␻ product aturally occurring plasmids are usually stably maintained in represents a new element involved in the regulation of pSM19035 their bacterial hosts. This stability often must be accomplished N replication. We demonstrate both in vivo and in vitro that, in in spite of a very low number of plasmid copies per cell. Replication- ␻ control mechanisms play a very important role here by ensuring a addition to repressing its own synthesis, protein also blocks transcription from genes copS and ␦. Our observations indicate that constant number of plasmid copies per chromosome for segrega- ␻ tion to each daughter cell (reviewed in refs. 1–4). Additionally, the protein is a negative regulator linking plasmid copy control several specialized plasmid stabilization modes have been identi- to better-than-random segregation. fied. They can be divided into three major classes, a, b, and c. Class Materials and Methods a groups those functions that maximize the number of plasmid units ␣ to be segregated between the daughter cells (reviewed in refs. 4–6). Bacterial Strains and Plasmids. Escherichia coli strains DH5 and In class b, the plasmid encodes a toxin that kills the plasmid-free BL21(DE3) and Bacillus subtilis strains YB886 and YB1015 (recA4) progeny and an antitoxin that neutralizes its cognate toxin or were used (13, 20). Plasmids pDB101, pBT233, pBT291, pBT346, prevents its synthesis (7). Class c includes active plasmid partition pBT312, and pBT347 (10, 13, 14), pBT338 (21), and pBT205 (S. systems that ensure the localization of at least one plasmid molecule Chai and J.C.A. unpublished results) were used. The plasmid-borne at the site of future septal growth (cell-quarter site) of the daughter Pcop (pUC57, coordinates 18976 to 64 of pDB101), P␦ (pBT291, cells (8, 9). The class b and c systems have been described for several 4735 to 5635), and P␻ (pCB30, 6377 to 6754) were used for plasmids of Gram-negative bacteria, including P1, F, RK2, and R1 promoter analysis. Plasmid pRC1 was constructed by in vitro (1–4, 7). The mechanisms used by plasmids of Gram-positive deletions of pDB101 and is composed of the copS, repS, and erm bacteria to ensure that each cell receives at least one plasmid copy genes (coordinates 16738-2546 and 7824–9622). The plasmid-borne at cell division (better-than-random segregation) are poorly under- ␦ and ␻ genes (pBT346–1, coordinates 4685–6754), ␻ and ␧ genes stood. (pBT346–2, 5800–7081), ␻ gene (pBT346–3 and pT7-␻, 6312– The low-copy-number and broad-host-range plasmids of the 6894), and ␧ gene (pBT346–4, 6595–7081) were constructed by in ␤ inc18 family (pIP501, pAM 1, and pSM19035) from Gram-positive vitro deletions of pBT346. Plasmid pBT346–5, which is derived from Ͼ bacteria share an extensive sequence identity ( 90%) in their pBT346–3, contains a STOP TAA codon at the sixth triplet of the replication (Cop, RNA III, Rep, ori;Fig.1A) and stability (SegA ␻ gene. The 5Ј noncoding region of copS (18560 to 1), ␦ (4695 to and SegB) regions (10–12). pSM19035 has extraordinarily long ␻ ␧ Ϸ 5800), (6312 to 6522), and (6529 to 6899) genes were fused to inverted repeated sequences that comprise 80% of the plasmid the promoterless lacZ gene of E. coli plasmid pBT205. molecule. The sequences required to ensure plasmid replication and its stable maintenance are localized within the inversely re- peated segments (13, 14), which means that they are duplicated in Abbreviation: EMSA, electrophoretic mobility-shift assay. the plasmid. Replication is controlled by two negative regulators, ‡To whom reprint requests should be addressed. E-mail: [email protected]. each of which reduces the amounts of mRNA coding for the The publication costs of this article were defrayed in part by page charge payment. This initiation protein, Rep. One of them, Cop, represses transcription article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. of the rep mRNA, whereas the other one is a stable antisense RNA §1734 solely to indicate this fact. 728–733 ͉ PNAS ͉ January 18, 2000 ͉ vol. 97 ͉ no. 2 Downloaded by guest on September 28, 2021 Fig. 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, P␦, P␻, and P␧) 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, ␦, ␻, ␧, and ␨) 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), P␦, and P␻promoters. The transcription start sites of Pcop1, Pcop2, and P␦ and P␻, determined by primer extension assays, are denoted by bent arrows. Conserved Ϫ35 and Ϫ10 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. Bacterial Growth, Transformation, and Plasmid Copy Number. B. Molecular Mass Determination. Gel filtration chromatography was subtilis and E. coli cells were grown and made competent as carried out in 50 mM Tris⅐HCl, pH 7.5͞150 mM NaCl at 4°C with ͞ described previously (20). The promoterless lacZ and its derivatives aflowrateof0.5ml min, and the A280 was measured. Ten and (cop:lacZ, ␦:lacZ, ␻:lacZ, and ␧:lacZ fusions), present in pBT205, twenty micrograms of ␻ protein (3 and 6 nmol) was applied onto were integrated by a double-crossover into the amyE locus of the Superose 12 column (Pharmacia). A standard curve of Kav vs. YB886 strain. In a second step, the recA4 mutation was introduced log of molecular masses was determined as recommended by into the background. The ␻ gene (6312–6727) was integrated via Pharmacia. Equilibrium centrifugation of ␻ protein (2, 4, and 6 ␮ ⅐ ͞ plasmid pBT205 into the chromosome of YB886 to construct strain M) in 25 mM Tris HCl, pH 7.5 150 mM NaCl was analyzed in a YB886-␻. Beckman Optima XL-A (Beckman Coulter) operating at 8,000 and YB886 and YB886-␻ strains bearing plasmid pRC1 were grown 15,000 rpm (An-Ti60 rotor) for 12 hr at 20°C. to the middle exponential phase, and plasmid DNA from 5-ml ϭ Primer Extension Assays. Transcription reactions were performed in cultures (OD650 0.8) was isolated. The relative plasmid copy a 25-␮l final volume in buffer B [25 mM Tris⅐HCl,pH7.5͞10 mM number was obtained by comparing the intensity of the ethidium ͞ ͞ ͞ ͞ bromide-stained plasmid DNA bands (HindIII cleavage) with MgCl2 80 mM KCl 5mM(NH4)2SO4 2% glycerol 12 mM NaCl] containing ATP, CTP, GTP, and UTP (200 ␮M of each), 2 IMAGE QUANT (Molecular Dynamics) software.