Proc. Natl. Acad. Sci. USA Vol. 83, pp. 9645-9649, December 1986 Genetics Positive and negative roles of an initiator protein at an origin of replication (plasmid R6K/promoter deletions/replication control/autoregulation/immunoassays) MARCIN FILUTOWICZ, MICHAEL J. MCEACHERN, AND DONALD R. HELINSKI Department of Biology, University of California, San Diego, La Jolla, CA 92093 Contributed by Donald R. Helinski, September 5, 1986 ABSTRACT The properties of mutants in the pir gene of origin activity (20) and autogenous regulation of the pir gene, plasmid R6K have suggested that the a protein plays a dual respectively (18, 21). role; it is required for replication to occur and also plays a role A negative role for the irprotein in the control ofR6K copy in the negative control of the plasmid copy number. In our number was suggested originally by the isolation of a mutant present study, we have found that the Xr level in cell extracts of of the R6K derivative plasmid pRK419, designated Cos405, Escherichia coli strains containing R6K derivatives is surpris- that maintained itself at a greatly increased copy number as ingly high (-104 dimers per cell) and that this level is not a result of a single amino acid substitution within the Xr altered in cells carrying high copy number pir mutants. The protein. This mutational change was recessive to wild-type wild-type and a high copy mutant (Cos4O5) pir gene were protein (22). The properties ofthis mutant protein along with inserted downstream of promoters of different strengths to the well-established positive function of ir protein in R6K measure the copy number of an R6K y replicon as a function replication (3, 4) lead to the conclusion that the ir protein ofa 1000-fold range ofintracellular ir concentrations. The data plays a dual role; it is both required for R6K replication and demonstrate that reducing the intracellular level of ii to 5% of yet is also capable of inhibiting it. its normal value can result in a substantial increase in copy This study examines the effect of varying ir concentration number of a y origin replicon and that a 17 level <1% ofnormal on plasmid copy number utilizing the wild-type pir gene and is still permissive for replication. Conversely, increasing the ii a copy-up mutant pir gene (Cos4O5) inserted downstream of level even a few-fold above normal results in a marked promoters of different strength. The varying levels of ir inhibition ofreplication ofplasmids containing a single, two, or protein in E. coli carrying these constructs were determined all three of the R6K origins (a, i, and y). We have also shown by an immunological assay using antibody preparations that the replication inhibition mediated by excess ir is greatly raised against purified ir protein. The data clearly indicate reduced by thepir4O5 Cos mutation. These results demonstrate that there is not a linear relationship between Xfconcentration that the total level of ir protein is not rate-limiting for a Y and plasmid copy number and that excessive levels ofirresult replicon. We have also determined the sensitivity ofthepir gene in a substantial reduction in plasmid copy number. Our promoter to a wide range of ir concentrations. The activity of studies also suggest that the major role of autorepression of this promoter is stimulated by very low ir levels and is almost ir expression in the R6K system is to prevent the i protein entirely inhibited when the protein is overproduced 2-fold. from accumulating to levels high enough to inhibit plasmid replication. The self-transmissible antibiotic resistance plasmid R6K of Escherichia coli is a member of a large group of bacterial MATERIALS AND METHODS plasmid replicons that share common features, including nucleotide sequence repeats at their replication origin and in Bacterial Strains and Plasmids. The E. coli strains C600, several cases an autoregulated initiator protein that binds to MC1000 (21), C2100 (20), MB2 (15), P678-54 (2), YSlkpir*, these repeats (1, 2). Replication functions ofplasmid R6K are ll\pir*, 041 (23), and 451(24) were used in the experiments clustered within a 4-kilobase-pair (kbp) segment (3-6). This indicated in the figure legends. Characteristics of plasmids region contains three replication origins designated a, 13, and R6K (25), pRK35 (24), pRK419 and pRK526 (3), pMF26 (15), y(7, 8) and the structural genespir (9) and bis (10) that encode and of other plasmids used in this study are indicated in the for the IT and bis proteins, respectively. The 35-kDa iT protein text or will be described elsewhere. (9) is required for activity ofall three origins (3, 11, 12), while Preparation of Lysates for Protein and DNA Electrophore- the requirement for the 17-kDa bis protein appears to be sis. Total cell lysates from 1- or 1.5-ml cultures were prepared restricted to the /3 origin of replication (10, 13). The minimal for protein analysis according to Laemmli (26). Analysis of genetic information that is required for stable maintenance of cellular DNA content in 0.6% agarose gels was carried out this plasmid at its copy number of -15 per chromosome with total lysates prepared from 1 ml of cultures in logarith- equivalent consists of the -400-bp y origin (14) and the pir mic phase. Cells were harvested at room temperature, gene, whose product can function when supplied in trans (3, washed with buffer A (150 mM NaCl/100 mM Na2EDTA/10 11). The Ir protein purified in its native dimeric form (15), or mM Tris'HCl, pH 10.2) and resuspended in 0.1 ml ofthe same as a hybrid with a (B-galactosidase (16) or collagen (17) buffer. The cell suspension was treated with 10 1.d of moiety, has been shown to bind to the seven 22-bp repeats in lysozyme (5 mg/ml; Sigma), incubated for 10 min at 370C the R6K y origin and to an eighth 22-bp repeat and a smaller followed by the addition of 2.5 Al of 10% NaDodSO4 inverted pair of repeats that are present in the operator-pro- (Bio-Rad) and 10 A.l of Pronase (20 mg/ml), and incubated at moter region of the pir gene (15, 16, 18, 19). Binding of the IT 370C for 2 hr. The mixture was then incubated for 30 min at protein to these two regions appears to be essential for y 650C and after cooling to room temperature, 40 ttl of solution B (10% urea/3% Ficoll/0.02% xylene cyanol/0.02% bromo- The publication costs of this article were defrayed in part by page charge phenol blue) was added. Chromosomal DNA was sheered by payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: bp, base pair(s). 9645 Downloaded by guest on September 30, 2021 QKA6 Genetics: Fflutowicz et al. Proc. Natl. Acad. Sci. USA 83 (1986) 1 min of mixing in a Vortex followed by vigorous passing of DNA replication is exhibited within a particular range of ir lysate through a 200-,ul Pipetman tip. Samples were frozen at concentrations. To test this, plasmids that overproduce -200C, thawed, and 1-to 20-1ulaliquots were electrophoresed either the wild-type or a mutant ir protein were constructed. under the conditions described in the figure legends. The plasmid pairs pPT20, pPT21 and pPT32, pPT39 contain DNA and Protein Blotting. Southern analysis was carried a FnudII-pir segment from the mutant pir4O5-Cos (22) and out as described (27) with the probes described in the figure wild-type plasmids, respectively, cloned into the pBR322- legends. Autoradiographs were traced with an LKB Ultro- derived vector pKJB825 (15). In one orientation (pPT20 and scan XL laser densitometer. Plasmid copy number was pPT39) the pir gene is under the control of a conditional determined by using an extract from strain YSI-Xpir* con- promoter (XPR), while in the opposite orientation (pPT21 and taining plasmid pRK526 as the control. This strain contains pPT32) it is under the control of a constitutive vector a single copy of a y-origin fragment inserted into the chro- promoter. The level of Xr protein in the E. coli host P678-54 mosome (3), and the copy number ofthe pRK526 plasmid was containing each of these plasmids was determined by estimated to be 15 per chromosome equivalent. The transfer Coomassie blue staining and immunoblotting analyses. As of proteins after NaDodSO4/PAGE was carried out as shown in Fig. 1A, plasmids with the constitutive promoter described (28). After transfer, the nitrocellulose was washed upstream of the pir gene overproduce the ir protein at 30'C. three times with TBS buffer (150 mM NaCl/20 mM Tris'HCl, A further increase in the cellular level of r can be seen in cell pH 7.5) and incubated at room temperature overnight with a cultures carrying plasmids pPT20 and pPT39 when shifted to purified ir IgG preparation in TBS buffer containing 1% 370C. Since the stained gel procedure did not allow for bovine serum albumin (fraction V; Sigma). The nitrocellulose quantitative determination of the amount of ir, the level of was washed three times with TBS buffer and incubated for 1 this protein in samples containing plasmids pPT32, pPT39, hr at 370C with a peroxidase-conjugated fraction of goat and pRK419 was determined by immunostaining (Fig. 1B). anti-rabbit IgGs at a concentration recommended by the Clones containing pPT39 and pPT32 produce about 2-fold and supplier (Cooper Biomedical, Malvern, PA). The immuno- 8-fold more ir protein, respectively, than those containing the blot was developed as described (10). R6K derivative plasmid pRK419.