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Copy-Up Mutants of the Plasmid RK2 Replication Initiation Protein

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Copy-Up Mutants of the Plasmid RK2 Replication Initiation Protein Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3559-3564, April 1996 Biochemistry Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins (direct repeats/regulation of DNA replication/DNA-protein interaction/antibiotic resistance/Escherichia coli) ALESSANDRA BLASINA*, BARBARA L. KITTELL*, ARESA E. TOUKDARIAN, AND DONALD R. HELINSKIt Center for Molecular Genetics and Department of Biology, University of California, San Diego, La Jolla, CA 92093-0634 Contributed by Donald R. Helinski, December 19, 1995 ABSTRACT The broad host range plasmid RK2 repli- characteristic of a major class of plasmids in Gram-negative cates and regulates its copy number in a wide range of bacteria (for review, see ref. 10). Gram-negative bacteria. The plasmid-encoded trans-acting The trfA gene of RK2 encodes two replication initiation replication protein TrfA and the origin of replication oriV are proteins of 44 kDa (TrfA-44) and 33 kDa (TrfA-33) as a result sufficient for controlled replication of the plasmid in all of an internal translational start in the same open reading Gram-negative bacteria tested. The TrfA protein binds spe- frame (11-13). Either protein alone is sufficient for RK2 cifically to direct repeat sequences (iterons) at the origin of replication in E. coli and a wide range of Gram-negative replication. A replication control model, designated handcuff- bacteria, with the exception ofPseudomonas aeruginosa, which ing or coupling, has been proposed whereby the formation of specifically requires TrfA-44 (14-16). coupled TrfA-oriV complexes between plasmid molecules re- The finding that the copy number of intact RK2 or of RK2 sults in hindrance of origin activity and, consequently, a minireplicons is not increased by increasing the concentration shut-down of plasmid replication under conditions of higher of the TrfA initiation protein over a 170-fold range in vivo (17) than normal copy number. Therefore, according to this model, and the demonstration that RK2 iterons specifically inhibit the coupling activity of an initiation protein is essential for copy replication of an RK2 replicon in vivo or in vitro even in the number control and a copy-up initiation protein mutant should presence of excess amounts of the initiation protein (18) gave have reduced ability to form coupled complexes. To test this rise to a model of replication control that involves a TrfA- model for plasmid RK2, two previously characterized copy-up mediated association of plasmid molecules at their origin of TrfA mutations, trfA-254D and trfA-267L, were combined and replication (18, 19). This handcuffing or coupling model the resulting- copy-up double mutant TrfA protein TrfA- proposes that at intracellular concentrations of plasmid mol- 254D/267L was characterized. Despite initiating runaway (un- ecules at or above the average copy number, all of the controlled) replication in vivo, the copy-up double-mutant TrfA TrfA-bound plasmid molecules are reversibly coupled at their protein exhibited replication kinetics similar to the wild-type origins resulting in the steric hindrance of origin activity. protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA- Subsequently, as a result of cell growth, the plasmid concen- 254D/267L proteins were then examined for binding to the tration decreases and the coupled complexes dissociate, re- iterons and for coupling activity using an in vitro ligase-catalyzed leasing origins active for the initiation of DNA replication. multimerization assay. It was found that both single and double Essentially the same model was proposed earlier for the TrfA mutant proteins exhibited substantially reduced (single regulation of the iteron-containing plasmids P1 and R6K (20, mutants) or barely detectable (double mutant) levels ofcoupling 21). In the case of both of these plasmids, and consistent with activity while not being diminished in their capacity to bind to the the coupling model, the plasmid-encoded initiation protein has origin of replication. These observations provide direct evidence been shown in vitro to specifically complex DNA fragments in support of the coupling model of replication control. containing the specific origin of replication. Initiation protein mutants that regulate replication at an increased copy number The stable maintenance of a plasmid in a bacterial cell is in (copy-up mutants) have been isolated for a number of iteron- large part due to plasmid-encoded factors that regulate the containing plasmids (22-29). In the case of RK2, the finding copy number of the extrachromosomal element in its host. that replication of minireplicons by a copy-up TrfA mutant is RK2 differs from most other plasmids by its ability to initiate much less sensitive to iteron inhibition than replication by and regulate its replication in a wide range of Gram-negative wild-type TrfA both in vivo and in vitro has provided additional bacteria (1). Maintenance of this 60-kb plasmid at a copy evidence in support of the coupling model (18). number of four to seven copies per chromosome in Escherichia A key prediction of the coupling model is that a defect in the coli is dependent on a specific origin of replication, oriV, and ability of the replication initiation protein to intermolecularly the plasmid-encoded trans-acting replication initiation protein couple iterons, while not affecting the ability of the protein to TrfA (2-5). The TrfA protein and the oriVsequence have been bind to the iterons and initiate replication, would lead to an shown to be sufficient for the initiation and control of repli- increase in plasmid copy number. In this study two previously cation of RK2 in almost all Gram-negative bacteria examined isolated copy-up trfA single point mutations were combined to date (6). A major feature of the nucleotide sequence of RK2 within a trfA gene. It was observed that introducing both oriV is the presence of eight 17-bp direct repeats (iterons) copy-up mutations into trfA resulted in a protein that initiated arranged in two clusters, one with three iterons the other with "runaway" replication of RK2 oriV in E. coli while exhibiting five iterons (7). Deletion of the upstream three-iteron cluster replication kinetics similar to wild-type TrfA protein in vitro. does not inactivate the origin in E. coli but does result in an When the purified wild-type and mutant forms of the TrfA increase in copy number and some instability (8, 9). The protein were tested in vitro for binding to the RK2 origin and presence of multiple iterons at the origin of replication that are for intermolecular coupling, it was found that all three mutant bound by a plasmid-encoded replication initiation protein is proteins bound as well or better to the iterons at the origin but were substantially diminished (either single mutant) or exhib- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in *B.L.K. and A.B. contributed equally to this work. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3559 Downloaded by guest on September 24, 2021 3560 Bohmsr:Baiae l Proc. Natl. Acad. Sci. USA 93 (1996) ited barely detectable levels (double mutant) in their ability to gyrA thi hsdR17 (rk-mk) supE44 relA A(lac-proAB) F'traD36 couple TrfA-bound origins. These findings satisfy a key pre- proAB laclIZ AM15] was used. diction of the coupling model for the regulation of plasmid The recessiveness of TrfA-33(254D/267L) to TrfA-33 was RK2 replication. determined by using pAL100 and pAL102, penicillin-resistant RSF1010O-based constructs that express -4- and -44-fold the MATERIALS AND METHODS level of TrfA, respectively, expressed by RK2 (17). Expression and Purification of TrfA and His6-TrfA Wild- Construction of trfA(254D/267L) by Site-Directed Mu- Type and Mutant Proteins. Plasmid pBK3 was constructed to tagenesis. Two copy-up trfA mutations, 254D and 267L (22), enhance TrfA-33 expression from pKK223-3 (Pharmacia). were combined by site-directed mutagenesis using the Altered The 0.9-kb EcoRI-Pst I trfA-33 sequence from pRD10-16 Sites in vitro mutagenesis system from Promega. The 1.2-kb was inserted into the same sites of pUC19. This plasmid was EcoRI-Pst I fragment from pRD110-34:267L containing the linearized with EcoRI, subjected to BAL-31 nuclease treat- trfA gene with the 267L mutation was inserted into the ment, ligated to EcoRI linkers, and then digested with Pst I. EcoRI-Pst I sites of pSELECT to generate pSELECT- The EcoRI-Pst I fragments were then inserted into the same trfA(267L). The oligonucleotide 5'-ACAGGCGACGGC- sites of pKK223-3. Transformants were analyzed for increased CATGGACTTCACGTCCGA-3' was used to introduce the TrfA expression by using a Western slot blot analysis. The 254D mutation (underlined A) and a Sty I site (underlined C; construct expressing the highest levels of TrfA, designated resulting in no alteration of the protein sequence) into pSE- pBK3, was sequenced to determine the extent of the deletion. LECT-trfA(267L). Site-directed mutagenesis was performed An Sfi I-Pst I fragment containing part of the trfA(254D/ by following the manufacturer's protocol. After strand synthesis 267L) gene was isolated from pBK100 and inserted in place of and ligation, the reaction mixture was used to transform E. coli the Sfi I-Pst I fragment containing the trfA-33 gene in pBK3. TGI [A(lac-proAB) (F' lacI proAB lacZ AM15)]. More than 5000 The resulting construct, pBK3-254D/267L, was established in penicillin-resistant colonies were obtained, -80% of which had E. coli BB3 [(F' proAB lacIqZ AM15 TcR) recA endA gyrA thi acquired a new Sty I site and presumably also carried the 254D hsdR17 supE44 relA lac-] for purification of the TrfA- mutation.
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