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Stability of Cole1-Like and Pbr322-Like Plasmids in Escherichia Coli

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Stability of Cole1-Like and Pbr322-Like Plasmids in Escherichia Coli Molecular Microbiology (1989) 3(12). 1745-1752 Stability of ColE1-like and pBR322-like plasmids in Escherichia coli J. Aya la-San martin and M. C. G6mez-Eichelmann* piasmid-specifio DNA sequence to a cellular partition Departamento de Biologia Molecular, Instituto de system (Miki ef al., 1980; Gustafsson et al., 1983); (iii) Investigaciones Biomedicas, Universidad Nacional site-specific recombination of multimers to provide an Autonoma de Mexico. Apto Postal 70-228, 04510 adequate number of units for partitioning (Austin et al.. Mexico. D.F. Mexico. 1981; Sherratt et aL, 1986); (iv) post-segregational killing. which prevents the appearance of plasmid-free cells in a growing culture (Jaffe et al.. 1985; Gerdes etal., 1986); and Summary (iv) temporal inhibition of cell division if the plasmid The average copy number, the level of ampicillin replication process does not provide a sufficient number resistance conferred by one plasmid, and the degree of plasmid copies per cell cycle (Ogura and Hiraga, 1983a; of plasmid multimerization were determined for Miki ef a/., 1984). Different plasmids display distinct several ColE1-like and pBR322-like plasmids. From strategies to ensure an accurate partition and therefore the results obtained, the variance of the units of stability. The low oopy-number plasmids, F. R1 and partition corresponding to each plasmid studied was prophage P1, and the intermediate copy-number plasmid, calculated. Experimentally determined plasmid stabi- pSCIOI, in addition to other strategies to ensure their lity was compared with that calculated using the stability, code for functions involved in active partition variance of the units of partition and the ratio between (Meacock and Cohen, 1980; Nordstrom ef al.. 1980; the generation times of plasmid-free and of plasmid- Austin and Abeles, 1983; Gustafsson ef ai. 1983; Ogura carrying cells, assuming that the units of partition are and Hiraga, 1983b). However, there are no reports of distributed randomly between daughter cells. active partition mechanisms for high copy-number plas- Stability of the pBR322-like plasmids present mainly mids. The stability of plasmids is a complex phenomenon; as monomers in the bacterial host was consistent with however, as has been suggested by Summers and random partitioning, whereas pBR322-like plasmids, Sherratt (1984), bacterial cells carrying high copy-number present mainly as dimers, and the ColE1-like plasmid plasmids with no active partition mechanism can generate showed greater stability than that predicted with plasmid-free cells if the number of units of partition in random partitioning at cell division. some cells close to division is relatively low. The present study was designed to experimentally test the hypothesis that partition of high copy-number plas- mids is at random. For this purpose, a bacterial population Introduction was considered to be formed by various cell sub-popu- Plasmids are extrachromosomal DNA replicons present in lations carrying different numbers of units of partition and many bacteria in a defined number of copies per cell. For therefore showing different probabilities for generation of some plasmids, this number may be as low as one to six, plasmid-free cells. It was also considered that in a whereas for others it may be as high as 100. Many bacterial population growing without selective pressure, low-copy and multi-copy plasmids are stably maintained the variance of units of partition remains constant after in a bacterial population after many generations of growth reaching an equilibrium. If this hypothesis is correct, the under non-selective conditions; however, most of the stability of these plasmids should be a function of the multi-copy plasmlds constructed in vitro and used as variance of the number of units of partition in cells near cloning vectors are unstable and are lost from cultures at division. frequencies ranging from 10"^ to 10"^ per cell per Plasmids having different transcriptionai pattems as generation (Summers and Sherratt, 1984). well as rop* and rop~ plasmids were selected. The Five main mechanisms for ensuring plasmid stability plasmid transcriptional pattern can modify the degree of have been proposed; (i) a precise replication control plasmid multimerization and, therefore, plasmid stability (Davison, 1984; Lacatena et al., 1984); (ii) an active (Bujard ef al., 1985). The rop gene encodes for a protein partition mechanism mediated by the attachment of a that represses plasmid replication (Laoatena etal., 1984) and probably determines a broader piasmid copy-number Received 2 July. 1989. *For correspondence. TeJ. (5) 5503893. variance (Summers and Sherratt, 1985). 1746 J. Ayala-Sanmarti'n and M. C. Gomez-Eichelmann Piasmid copy number, piasmid multimerization, and was calculated from the distribution of single-cell resist- single-cell resistance to ampicillin were determined in ance to different concentrations of ampicillin (Fig. 1). From strain C600 carrying each of the plasmids tested. From the these data, the average level of ampicillin resistance in the results obtained, the variance of the units of partition was bacterial population was calculated. The average level of calculated. Piasmid stability was experimentally resistance to ampicillin and the average piasmid copy determined and compared with that calculated using the number were used to determine the resistance level to experimentally obtained values for variance of the units of ampicillin conferred by one piasmid (Table 1). Piasmid pT7 partition. conferred a higher resistance to ampicillin than that conferred by the other plasmids tested. This higher resistance level is probably due to the presence in pT7 of a Results stronger bla promoter or to a higher degree of transcrip- tional readthrough into this gene from distal promoters. Average piasmid copy number and level of ampicillin The ampicillin resistance conferred by one copy of the resistance conferred by one piasmid derivative plasmids plOI or pBR329 was lower than that conferred by the original plasmids pBR322 or pBR327, The average piasmid copy number per cell determined by respectively. As mentioned before, the former plasmids a method which yields an accurate piasmid to chromo- contained an insertion of 877bp in the unique EcoRI site some ratio (Womble ef a/., 1977) was 11.5 for C0IEI present in both pBR322 and pBR327. This insert sepa- (average of two independent experiments) and 11 for pT7 rates the anti-tet promoter (Covarrubias and Bolivar, 1982) (average cf three experiments) (Table 1). The copy from the bla gene; therefore, it is possible that in plOI and numbers for plasmids pBR322, plO1, pBR327 and pBR329 the number of transcripts containing the bla gene pBR329 were determined by fluorescence densitometry is lower than that in pBR322 and pBR327. (Projan etal., 1983) using piasmid pT7 as control. With this method, the average copy number for piasmid pBR322 was 61; for plOI, 37; for pBR327. 74; and for pBR329, 20 Piasmid multimerization (Table 1). The ccpy number for piasmid pBR327 (rop~) was, as expected, higher than that for pBR322 {rop^) The degree of piasmid multimerization for the plasmids (Lacatena et al., 1984). However, the copy number for tested is shown in Table 2. Multimerization of piasmid plasmids plOI and pBR329 which carry the rep region of plOI was calculated by both electron microscopy and pBR322 and pBR327, respectively, was lower than the densitometry, as described in the Experimental pro- number expected. The main difference between pBR322 cedures. The results obtained with these two methods and pBR327 and the derivative plasmids plOI and were similar; therefore piasmid multimerization for the pBR329 is the insertion in the EcoRI site of the former other plasmids was determined using only the second plasmids of a fragment of 877 bp carrying the gene cat method. The degrees of multimerization for C0IEI and pT7 (Experimental procedures; Covarrublas and Bolivar, were similar. The relatively high percentage of dimers 1982). presented by these two plasmids was unexpected. C0IEI Since there is a linear correlation between resistance to encodes a determinant, cer, which converts multimers to ampicillin and gene dosage (Uhlin and Nordstrom, 1977). monomers (Summers and Sherratt, 1984); pT7 carries the the level of ampicillin resistance conferred by one piasmid resolution system encoded by Tn? (Arthur and Sherratt, 1979) in addition to the cer determinant. The prevalent molecular form of plasmids pBR322 and pBR327 was the Table 1. Piasmid copy number and level of ampjcillin resistance conferred by one piasmid to strain 0600. dimer; meanwhile plO1 and pBR329 were present mainly as monomers. Average ampicillin Ampicillin Average piasmid resistartce resistance Piasmid copy number/cell" (fig ml 'f (iig mlVcop/ Distribution of piasmid copy number in the bacterial PT7 11 2300 210 pBR322 61 7400 120 population at different times plOl 37 1600 .#.: pBR327 74 6700 90 The distribution of piasmid copy number in cells growing pBR329 20 900 45 exponentially under non-selective pressure at different a. The copy numbers were estimated by determination of CCC DNA (pT7) times was studied. These distributions were determined in and by fluorescence densitomelry (pBR322, plOl. pBR327, and pBR329). a cell culture started with ceils from Luria plates containing b. Calculated from histograms, as shown in Fig. 2. The histograms were a relatively high concentration of Tc (50^.g ml"^). The obtained from experiments represented as in Fig. 1. c. Ratio between
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