Journal of Biorechnology. 18 (1991) 0 1991 Elsevier Science Publishers ADONIS 016816569100078L
BIOTEC
141-152 B.V. 0168-1656/91/$03.50
141
00594
The effect of the partition locus on plasmid stability and expression of a prolonged chemostat culture Allison Deportmenr
E. Weber, Peng Yu and Ka-Yiu
oj Chemical (Received
Engineering,
9 May
1990;
Rice
revision
University. accepted
Houston, 9 September
San Texas,
U.S.A.
1990)
Summary The stability, copy number, and gene expression of the pBR322 plasmid containing the par-locus under prolonged cultivation were studied. In the initial stage of the experiment it was observed that the par-locus had a stabilization effect on plasmid maintenance. This observation was consistent with previously reported results. However, after approximately 225 h, ,a mixed population of plasmid-containing and plasmid-free cells appeared. The mixed culture was stably maintained for approximately 200 h. In addition, the relative plasmid copy number showed an increase as compared to the par- culture. After 100 h the copy number decreased, reached a minimum, then stabilized. The p-lactamase activity, was not significantly affected by the par-locus. Plasmid stability;
Par-locus; Prolonged cultivation
Introduction The stable maintenance of a plasmid in a growing culture depends on two primary factors: the faithful replication of the plasmid and the accurate segregation of the plasmid during cell division. If a mechanism governing the accurate partitioning of the plasmid is not present the distribution of the plasmid in the daughter cells is presumed to occur randomly (Scott, 1984). There is evidence, however, that on certain plasmids mechanisms exist which guarantee a more accurate partitioning of
Correspondence U.S.A.
lo: K.-Y.
San. Dept.
of Chemical
Engineering,
Rice
University,
Houston,
TX 77251-1892,
142
the plasmid set during cell division (Miki et al., 1980; Nordstrom et al., 1980). The observed stabilization effect is more apparent in cultures where low copy number plasmids are stably inherited (Jacob et al., 1963; Nordstrom et al., 1984a, b). The presence of a specific DNA sequence that assures stable plasmid inheritance in a growing cell population has been observed in many laboratories. The region affecting the partitioning of the plasmid has been identified in the Clo DF13 (Hakkaart et al., 1982a, b; 1984), Ti (Gallie et al., 1985) and ColEl plasmid (Skogman et al., 1983). Systems involved in plasmid segregation have also been found in low copy number plasmids Pl (Austin et al., 1981), F (Ogura and Hiraga, 1983), and the incFI1 plasmid, NRl (Miki et al., 1980). Two of the more studied partitioning systems are those on the Rl (Nordstrom et al., 1980) and pSClO1 plasmid (Meacock and Cohen, 1980). Many studies have been performed in which the stability region from a plasmid was cloned to plasmids lacking a stability factor (Boe et al., 1987; Lee and Edlin, 1985; Skogman et al., 1983; Zurita et al., 1984). These include the ParA and ParB genes from the Rl plasmid, ccd and sop stability region from the F plasmid, and the par-locus from the pSClO1 plasmid. In most cases, the stability of the plasmid had been increased. Four points concerning these experiments should be made. First, an increase in stability was a function of the stability factor inserted into the plasmid (Boe et al., 1987). Second, most of the experiments generally did not proceed longer than 160 generations. From previous experiments (Weber and San, 1988) it is believed this length of time is not adequate for long-term plasmid stability analysis. Third, the stability of some of the plasmids, after the addition of the par-locus, was not complete (Skogman et al., 1983). The stability’ function of the par-locus appeared to be dependent on the vector and the growth medium. In fact, it has been demonstrated that the par-locus does not stabilize all plasmids under all conditions and does not affect E. coli minichromosomes (Hinchliffe et al., 1981; Boe et al., 1987). Fourth, the effect of the par-locus on the cloned gene expression has not been reported. The focus of this work is to study the effect of the par function on the stability and productivity of the pBR322 plasmid under prolonged cultivation.
Material and Methods Bacterial strain and plasmid Escherichia coli RRl, a K-12 mutant (ATTC No. 31343), and the plasmid pBR322 (ATCC No. 37017) were used in this work. The plasmid carries a gene for the /3-lactamase enzyme. Construction of pBR322-par plasmid Plasmid pBR322-par pA37-par with EcoRI.
was constructed by first digesting both the pBR322 and Plasmid pA37-par was kindly supplied by Dr. Soberon
143
EcoRl
APR EcoRl
TCR
EcoRl
EcoRl Tq ligose
-5148
-2027 1584,330
bp
1904
%i 1330 lgo4 983
831
564 bp
D 4351
I__________________.pA37par I, 360 TCGTCTTCAAGAATTCCCCGGATCCGTCAGCTTGGGACAGTA.....CAGCGATTTGCCCGACGGAATCCGGG~~TTCTC AGCAGAAGTTCTTAAGGGGCCTAGGCAGTCGAACCCCTGTCAT.....GTCGCTAAACGGGCTGCCTTAGGCCCCTTAAGAG LpBR32d
II
1
IqBR322-->
Fig. 1. (A) Schematic showing construction strategy of the plasmid pBR322-par. (B) Verification of the plasmid by gel electrophoresis. Lane 1 is pBR322-par digested with Psrl and HirtdIII. Lane 2 is pBR322 digested with the same enzymes. Lane 3 is the molecular weight marker. (C) Agarose slab gel electropherogram of pBR322 and pBR322-par plasmid digested with Hinfl. Lanes 1 and 4 are the molecular weight marker. Lanes 2 and 3 are plasmid pBR322-par and pBR322 respectively. A 1631-bp band is missing in pBR322-par and is replaced by three new bands of 1248 bp, 696 bp and 90 bp (not visible) generated from pBR322-par. (D) Reconstructed nucleotide sequence of the junction between the par-locus and pBR322. The numbering within the par-locus follows that of Zurita et al. (see bp 360).
(Zurita et al., 1984). The digested DNA fragments were then allowed to self-ligate in the presence of T4 ligase. After transformation and screening of the ampicillin resistant colonies, clones containing the par-locus were isolated (Fig. 1). Media and culture condition The stock culture was maintained on Luria agar slants. Glucose and filter sterilized ampicillin and tetracycline (Sigma) were added at concentrations of 2 g
145
l-‘, 100 mg l-‘, and 25 mg l-‘, respectively. The reactor inoculum was grown in Luria Broth (LB) supplemented with glucose, ampicillin, and tetracycline at the appropriate concentrations. The medium was LB supplemented with 0.5 g 1-l glucose. Detailed descriptions of the process conditions for the chemostat experiments are given in a previous article (Weber and San, 1989a). Two samples were taken daily from the bioreactor during the course of the experiments for plasmid stability analysis. Analysis of the plasmid content and enzyme activity were performed every two days. Plasmid stability analysis The percentage of plasmid-containing cells was determined by its ability to form colonies on ampicillin supplemented plates. The plates consisted of LB medium containing 2 g 1-l glucose, 15 g 1-l agar, and 100 mg 1-l ampicillin. Two methods were employed. In the first method, appropriately diluted samples from the reactor were spread evenly on LB agar plates, with and without ampicillin. Each plate was triplicated to reduce experimental error. The quantity of colonies formed on the plates, after 12 h of incubation at 37°C was compared and used to estimate the percentage of plasmid-containing cells in the culture. In the replica method, a sterile velvet pad was used to transfer 100-200 colonies from an LB plate to a plate containing 100 mg 1-l ampicillin. This procedure was repeated, using a different master plate, to increase the sample size. Transfer of individual colonies from the master plate to an antibiotic supplemented plate using sterile applicator sticks was performed as an additional control. Plasmid content and /Wactamase activity The determination of plasmid content and /3-lactamase activity follows similar procedures described earlier (Weber and San, 1989a). The copy number reported is per chromosomal DNA. P-Lactamase activity is defined as the quantity which hydrolyzes 1 pmol of benzylpenicillin per min at 37°C. The total protein concentration was determined by the Folin-Ciocalteu assay. An albumin and globulin solution (Sigma) was used as a protein standard.
Results
and Discussion
The stability of the pBR322-par plasmid is compared to a control experiment using the pBR322 plasmid (Weber and San, 1989b). The dilution rate for both experiments was maintained at 0.61 hh’. The results are shown in Fig. 2a. Initially, the addition of the par-locus results in a stabilization effect of the plasmid. The lag time, at which point plasmid-free cells were detected from the ampicillin supplemented plates, increased by approximately 50 h. In the next 100 h during which the portion of the plasmid-free cell increases, the behavior of the culture resembles that
Time
(h)
10 Time (h) Fig. 2. Comparison of the stability of a plasmid-containing culture for (a) the plasmid pBR322 (Cl) and plasmid pBR322-par (M) at D = 0.61 h-’ and (b) plasmid pBR322 (0) and plasmid pBR322-par (m) at D = 0.61 h-’ and the plasmid pBR322 under random cycling, D = 0.31 and 0.46 hK’ (v).
of the par- plasmid. It should be pointed out that literature data are seldom reported beyond this time (Skogman et al., 1983; Zurita et al., 1984). The chemostat culture, however, instead of showing a continual decline of the plasmid-containing population, as observed in the par- culture, reaches a pseudosteady-state where a mixed culture of plasmid-containing and plasrnid-free cells is stably maintained. During the period of 200-375 h the par+ culture demonstrates a very gradual decrease of the plasmid-containing cells. Within this time portion the percentage of plasmid-containing cells decreases from 63-38%. This ‘plateau’ is not maintained indefinitely and after 400 h into the experiment the plasmid-carrying population drops in a manner similar to previous experiments (Weber and San, 1988). Also shown in Fig. 2b are the results of another previously reported experiment in which the dilution rate was randomly cycled in the dilution rate, D = 0.31 and
147
0.46 h-’ (Weber and San, 1989a). It should be noted that the behavior demonstrated by the par+ plasmid resembles that of the plasmid-containing culture exposed to random oscillations. However, the mixed population in the random cycling experiment is maintained at a higher plateau, reflecting a higher percentage, approximately 60 to 70%, of plasmid-containing cells. In addition, the plateau persisted only for approximately 100 h, half the time period of the plateau observed with the par+ plasmid. In the random cycling experiment the stability was believed to be caused by the random perturbations of the dilution rate. An environment is thus created which is more advantageous to the plasmid-containing cells. As the culture with the par-locus was not exposed to any imposed oscillations a different mechanism is responsible for the stability of the culture. Two possible theories are explored. One possibility is due to plasmid conjugation. Similar results were predicted by a model describing a conjugative plasmid system (Esty, 1986). However, this is probably not the case as the plasmid pBR322 is not conjugative. In addition, there was no evidence of conjugation by the par+ plasmid in subsequent experiments. The presence of the par-locus is a more probable explanation. If it is assumed that a decrease in the plasmid-containing population is accompanied by a decrease in the plasmid copy number (Weber and San, 1990) a population with a lower plasmid copy number exists during the stable plateau. The par-locus, in turn, might be more efficient in evenly dividing the plasmids between the daughter cells at a lower copy number. This could also result in the observed stable mixed populations. The final decrease in the percentage of plasmid-containing cells is most likely due to the ‘ take-over’ of the plasmid-free cells and not a failure of the par-locus. Restriction enzyme patterns of the plasmid pBR322-par isolated from the later part of the experiment do not show any major structural changes (data not shown). The advantage of the par-locus is an increase in the accuracy of plasmid partitioning between two daughter cells during cell division. Based on this assump-
300
200 Time
Fig. 3. Percentage 50 (0). 100 (0).
(h)
of plasmid-containing cells that formed colonies on plates supplemented with 25 (0). and 25 mg 1-l tetracycline (v) for plasmid 500 (A), and 2000 (A) mg 1-l ampicillin pBR322-par at D = 0.61 h-‘.
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showing the percentage of plasmid-containing cells that formed colonies between a supplemented plates for pBR322-par at D = 0.61 h-’ from 90.5 to 259.25 h. (b) the percentage of plasmid-containing cells that formed colonies between a range of supplemented plates for pBR322-par at D = 0.61 hh’ from 283 to 450 h.
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tion, the par+ culture should be able to completely stabilize the culture. However, as shown in Fig. 2a, the addition of the par-locus to a plasmid does not necessarily ensure full stability. In addition, as seen in Figs. 3 and 4, the culture composition is
150
200
300
400
500
Time (h) Fig.
5. Comparison
of the specific plasmid pBR322
p-lactamase activity, based on the total protein (0) and plasmid pBR322-par (W) at D = 0.61 h-‘.
present,
for
rhe
not homogeneous as expected with respect to the copy number, but heterogeneous, similar to the par- systems (Weber and San, 1990). Sub-populations, based on the MIC study, can be observed after 100 h. This corresponds to the transient period when the plasmid-free cells begin to emerge. The behavior of the population distribution is also similar to the previous experiments (Fig. 4 a,b). In the par+ culture, however, the sub-populations are more evenly distributed during the stable plateau. A gradual shift of one sub-population to another is also observed. It was suggested that the sub-population reflects a heterogeneity of the plasmid copy number (Weber and San, 1990). The stable period, 200 to 375 h, consists of a large percentage of low copy number cells. This would agree with one of the assumptions proposed to explain the increase in stability of the par+ culture. The par-locus has been shown to increase the stability of many plasmids (Lee and Edlin, 1985; Skogman et al., 1983; Zurita et al., 1984). However, there have been no reports of its effect on plasmid copy number and expression. A comparison of the P-lactamase activity of the par+ and par- plasmids is shown in Fig. 5. In the first 250 h there is no noticeable difference between the two cultures. After this time the activity of the par+ culture increases slightly then gradually decreases. This trend corresponds to the period, up to 375 h, when the plasmids are stably maintained. In general, it would appear that the addition of the par-locus, except for prolonging the period of productivity, has no significant effect on the plasmid expression. Shown in Fig. 6 is the relative plasmid copy number, defined as the plasmid DNA per chromosome DNA. It can be seen that although the behavior is similar between the pBR322 and pBR322-par cultures, the population containing the par-locus has a higher relative plasmid copy number. This could be caused by the effective partitioning of the plasmids in the par+ cells resulting in a decrease in the net rate of plasmid loss. The increase in the lag time, Fig. 2a, also supports this
151
‘0 Time
(h)
Fig. 6. Comparison of the relative plasmid copy number, plasmid DNA per chromosomeDNA, for plasmidpBR322(0) and plasmidpBR322-par(U) a1D = 0.61 h-‘. assumption. The rapid decrease, from 100 to 200 h, in the plasmid content may be due to the appearance of the plasmid-free cells and a gradual loss of plasmid per cell. In summary, the addition of the par-locus to the pBR322 plasmid under prolonged cultivation proved beneficial. Both the stability and the plasmid copy number of the culture increased. Although there was no immediate effect on the P-lactamase activity, the long-term productivity increased. However, until the mechanism of the par function is better understood, the exact relationship between stability, plasmid copy number, and expression and the par-locus is not clear.
References Austin, S.. Ziese, M. and Sternberg, N. (1981) A novel role for site-specific recombination in maintenance of bacterial replicons. Cell 25, 729-736. Boe, R., Gerdes,K. and Molin, S.(1987)Effectsof genesexertinggrowth inhibition and plasmidstability on plasmid maintenance. .I. Bacterial. 169. 4646-4650. Esly, W.E. (1986) A model for fluctuation in the fraction of a bacterial population harboring plasmids. Theor. Popul. Biol. 30, 111-124. Gallie, D.R., Hagiya, M. and Kado, C.I. (1985) Analysis of Agrobocreriwn ftmli/ucie~u plasmid pTiC58 replication region with high-copy number derivative. J. Bacterial. 161, 1034-1041. Hakkaart. M.J.J., Vanden Elzen, P.J.M., Veltkamp. E. and Nijkamp, H.J.J. (1984) Evidence for sitespecific recomb’inant at pnrB. Cell 36, 203-209.
Hakkaart, M.J.J., Veltkamp, E. and Nijkamp, H.J.J. (1982a) Maintenance of the bacteriocinogenic plasmid Clo maintenance.
DF13 Mol.
in Escherichio coli cells, II. Specific Gen. Genet. 188, 338-344.
recombinant
functions
involved
in plasmid
Hakkaart, M.J.J., Wesseling,J.G., Veltkamp, E. and Nijkamp, H.J.J. (1982b) Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia co/i cells, 1. Localization and mutual interactions of four Clo DF13 incompatibility regions. Mol. Gen. Genet. 186, 531-539. Hinchliffe, E., Kuempel, P.L. and Masters, M. (1981) Escherichia co/i minichromosomes containing the pSClOI partitioning locus are not stably inherited. Plasmid 9, 286-297.
152
Jacob, F., Brenner. S. and Cuzin, F. (1963) On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28, 329-348. in pBR322 derivatives reduces the Lee, S.W. and Edlin, G. (1985) Expression of tetracycline resistan reproductive fitness of plasmid-containing Escherichia coti. Gene3 9, 173-180. Meacock. P. and Cohen, S.N. (1980) Partitioning of bacterial plasmids during cell division: a Cis-acting locus that accomplishes stable plasmid inheritance. Cell 20, 529-542. Miki, T., Easton, A.M. and Rownd. R.H. (1980) Cloning of replication, incompatibility and stability functions of R plasmid NRl. J. Bacterial. 141. 87-99. Nordstrom. K., Molin. S. and Aagaard-Hansen, H. (1980) Partitioning of plasmid Rl in Esclrerichia co/i I. Kinetics of loss of plasmid derivative deleted of the par region. Plasmid 4, 215-227. Nordstrom, K. and Aagaard-Hansen, H. (1984a) Maintenance of bacterial plasmids: comparison of theoretical calculations and experiments with plasmid Rl in Escherichia co/i. Mol. Gen. Genet. 197, Nordstrom, K., Molin, S. and Light, J. (1984b) Control of replication of the bacterial plasmids: genetics. molecular biology and physiology of the plasmid Rl system. Plasmid 12, 71-90. Ogura, T. and Hiraga, S. (1983) Partition mechanism of plasmid: IWO plasmid gene-encoded products and a Cis-acting region are involved in partition. Cell 32, 351-360. Scott, J.R. (1984) Regulation of plasmid replication. Microbial. Rev. 48, l-23. Skogman, G., Nilsson, J. and Gustafsson, P. (1983) The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia co/i. Gene 23, 105. Weber, A.E. and San, K.-Y. (1988) Enhanced plasmid maintenance in a CSTR upon square-wave oscillations in the dilution rate. Biotechnol. Lett. 10. 531-536. Weber. A.E. and San K.-Y. (1989a) Dynamics of plasmid maintenance in a CSTR upon square-wave perturbations in the dilution rate, Biotechnol. Bioeng. 34, 1104-1113. Weber. A.E. and San, K.-Y. (1989b) A comparison of two plating techniques to estimate plasmid stability of a prolonged chemostat culture. Biotechnol. Tech. 3, 397-400. Weber, A.E. and San, K.-Y. (1990) Population dynamics of a recombinant culture in a chemostat under prolonged cultivation. Biotechnol. Bioeng. 36, 727-736. Zurita, M., Bolivar, F. and Sober6n, X. (1984) Construction and characterization of new cloning vehicles VII. Construction of plasmid pBR327par. a completely sequenced stable derivative of pBR327 containing the par locus of pSC101. Gene 28, 119-122.
BIOTEC
141-152 B.V. 0168-1656/91/$03.50
141
00594
The effect of the partition locus on plasmid stability and expression of a prolonged chemostat culture Allison Deportmenr
E. Weber, Peng Yu and Ka-Yiu
oj Chemical (Received
Engineering,
9 May
1990;
Rice
revision
University. accepted
Houston, 9 September
San Texas,
U.S.A.
1990)
Summary The stability, copy number, and gene expression of the pBR322 plasmid containing the par-locus under prolonged cultivation were studied. In the initial stage of the experiment it was observed that the par-locus had a stabilization effect on plasmid maintenance. This observation was consistent with previously reported results. However, after approximately 225 h, ,a mixed population of plasmid-containing and plasmid-free cells appeared. The mixed culture was stably maintained for approximately 200 h. In addition, the relative plasmid copy number showed an increase as compared to the par- culture. After 100 h the copy number decreased, reached a minimum, then stabilized. The p-lactamase activity, was not significantly affected by the par-locus. Plasmid stability;
Par-locus; Prolonged cultivation
Introduction The stable maintenance of a plasmid in a growing culture depends on two primary factors: the faithful replication of the plasmid and the accurate segregation of the plasmid during cell division. If a mechanism governing the accurate partitioning of the plasmid is not present the distribution of the plasmid in the daughter cells is presumed to occur randomly (Scott, 1984). There is evidence, however, that on certain plasmids mechanisms exist which guarantee a more accurate partitioning of
Correspondence U.S.A.
lo: K.-Y.
San. Dept.
of Chemical
Engineering,
Rice
University,
Houston,
TX 77251-1892,
142
the plasmid set during cell division (Miki et al., 1980; Nordstrom et al., 1980). The observed stabilization effect is more apparent in cultures where low copy number plasmids are stably inherited (Jacob et al., 1963; Nordstrom et al., 1984a, b). The presence of a specific DNA sequence that assures stable plasmid inheritance in a growing cell population has been observed in many laboratories. The region affecting the partitioning of the plasmid has been identified in the Clo DF13 (Hakkaart et al., 1982a, b; 1984), Ti (Gallie et al., 1985) and ColEl plasmid (Skogman et al., 1983). Systems involved in plasmid segregation have also been found in low copy number plasmids Pl (Austin et al., 1981), F (Ogura and Hiraga, 1983), and the incFI1 plasmid, NRl (Miki et al., 1980). Two of the more studied partitioning systems are those on the Rl (Nordstrom et al., 1980) and pSClO1 plasmid (Meacock and Cohen, 1980). Many studies have been performed in which the stability region from a plasmid was cloned to plasmids lacking a stability factor (Boe et al., 1987; Lee and Edlin, 1985; Skogman et al., 1983; Zurita et al., 1984). These include the ParA and ParB genes from the Rl plasmid, ccd and sop stability region from the F plasmid, and the par-locus from the pSClO1 plasmid. In most cases, the stability of the plasmid had been increased. Four points concerning these experiments should be made. First, an increase in stability was a function of the stability factor inserted into the plasmid (Boe et al., 1987). Second, most of the experiments generally did not proceed longer than 160 generations. From previous experiments (Weber and San, 1988) it is believed this length of time is not adequate for long-term plasmid stability analysis. Third, the stability of some of the plasmids, after the addition of the par-locus, was not complete (Skogman et al., 1983). The stability’ function of the par-locus appeared to be dependent on the vector and the growth medium. In fact, it has been demonstrated that the par-locus does not stabilize all plasmids under all conditions and does not affect E. coli minichromosomes (Hinchliffe et al., 1981; Boe et al., 1987). Fourth, the effect of the par-locus on the cloned gene expression has not been reported. The focus of this work is to study the effect of the par function on the stability and productivity of the pBR322 plasmid under prolonged cultivation.
Material and Methods Bacterial strain and plasmid Escherichia coli RRl, a K-12 mutant (ATTC No. 31343), and the plasmid pBR322 (ATCC No. 37017) were used in this work. The plasmid carries a gene for the /3-lactamase enzyme. Construction of pBR322-par plasmid Plasmid pBR322-par pA37-par with EcoRI.
was constructed by first digesting both the pBR322 and Plasmid pA37-par was kindly supplied by Dr. Soberon
143
EcoRl
APR EcoRl
TCR
EcoRl
EcoRl Tq ligose
-5148
-2027 1584,330
bp
1904
%i 1330 lgo4 983
831
564 bp
D 4351
I__________________.pA37par I, 360 TCGTCTTCAAGAATTCCCCGGATCCGTCAGCTTGGGACAGTA.....CAGCGATTTGCCCGACGGAATCCGGG~~TTCTC AGCAGAAGTTCTTAAGGGGCCTAGGCAGTCGAACCCCTGTCAT.....GTCGCTAAACGGGCTGCCTTAGGCCCCTTAAGAG LpBR32d
II
1
IqBR322-->
Fig. 1. (A) Schematic showing construction strategy of the plasmid pBR322-par. (B) Verification of the plasmid by gel electrophoresis. Lane 1 is pBR322-par digested with Psrl and HirtdIII. Lane 2 is pBR322 digested with the same enzymes. Lane 3 is the molecular weight marker. (C) Agarose slab gel electropherogram of pBR322 and pBR322-par plasmid digested with Hinfl. Lanes 1 and 4 are the molecular weight marker. Lanes 2 and 3 are plasmid pBR322-par and pBR322 respectively. A 1631-bp band is missing in pBR322-par and is replaced by three new bands of 1248 bp, 696 bp and 90 bp (not visible) generated from pBR322-par. (D) Reconstructed nucleotide sequence of the junction between the par-locus and pBR322. The numbering within the par-locus follows that of Zurita et al. (see bp 360).
(Zurita et al., 1984). The digested DNA fragments were then allowed to self-ligate in the presence of T4 ligase. After transformation and screening of the ampicillin resistant colonies, clones containing the par-locus were isolated (Fig. 1). Media and culture condition The stock culture was maintained on Luria agar slants. Glucose and filter sterilized ampicillin and tetracycline (Sigma) were added at concentrations of 2 g
145
l-‘, 100 mg l-‘, and 25 mg l-‘, respectively. The reactor inoculum was grown in Luria Broth (LB) supplemented with glucose, ampicillin, and tetracycline at the appropriate concentrations. The medium was LB supplemented with 0.5 g 1-l glucose. Detailed descriptions of the process conditions for the chemostat experiments are given in a previous article (Weber and San, 1989a). Two samples were taken daily from the bioreactor during the course of the experiments for plasmid stability analysis. Analysis of the plasmid content and enzyme activity were performed every two days. Plasmid stability analysis The percentage of plasmid-containing cells was determined by its ability to form colonies on ampicillin supplemented plates. The plates consisted of LB medium containing 2 g 1-l glucose, 15 g 1-l agar, and 100 mg 1-l ampicillin. Two methods were employed. In the first method, appropriately diluted samples from the reactor were spread evenly on LB agar plates, with and without ampicillin. Each plate was triplicated to reduce experimental error. The quantity of colonies formed on the plates, after 12 h of incubation at 37°C was compared and used to estimate the percentage of plasmid-containing cells in the culture. In the replica method, a sterile velvet pad was used to transfer 100-200 colonies from an LB plate to a plate containing 100 mg 1-l ampicillin. This procedure was repeated, using a different master plate, to increase the sample size. Transfer of individual colonies from the master plate to an antibiotic supplemented plate using sterile applicator sticks was performed as an additional control. Plasmid content and /Wactamase activity The determination of plasmid content and /3-lactamase activity follows similar procedures described earlier (Weber and San, 1989a). The copy number reported is per chromosomal DNA. P-Lactamase activity is defined as the quantity which hydrolyzes 1 pmol of benzylpenicillin per min at 37°C. The total protein concentration was determined by the Folin-Ciocalteu assay. An albumin and globulin solution (Sigma) was used as a protein standard.
Results
and Discussion
The stability of the pBR322-par plasmid is compared to a control experiment using the pBR322 plasmid (Weber and San, 1989b). The dilution rate for both experiments was maintained at 0.61 hh’. The results are shown in Fig. 2a. Initially, the addition of the par-locus results in a stabilization effect of the plasmid. The lag time, at which point plasmid-free cells were detected from the ampicillin supplemented plates, increased by approximately 50 h. In the next 100 h during which the portion of the plasmid-free cell increases, the behavior of the culture resembles that
Time
(h)
10 Time (h) Fig. 2. Comparison of the stability of a plasmid-containing culture for (a) the plasmid pBR322 (Cl) and plasmid pBR322-par (M) at D = 0.61 h-’ and (b) plasmid pBR322 (0) and plasmid pBR322-par (m) at D = 0.61 h-’ and the plasmid pBR322 under random cycling, D = 0.31 and 0.46 hK’ (v).
of the par- plasmid. It should be pointed out that literature data are seldom reported beyond this time (Skogman et al., 1983; Zurita et al., 1984). The chemostat culture, however, instead of showing a continual decline of the plasmid-containing population, as observed in the par- culture, reaches a pseudosteady-state where a mixed culture of plasmid-containing and plasrnid-free cells is stably maintained. During the period of 200-375 h the par+ culture demonstrates a very gradual decrease of the plasmid-containing cells. Within this time portion the percentage of plasmid-containing cells decreases from 63-38%. This ‘plateau’ is not maintained indefinitely and after 400 h into the experiment the plasmid-carrying population drops in a manner similar to previous experiments (Weber and San, 1988). Also shown in Fig. 2b are the results of another previously reported experiment in which the dilution rate was randomly cycled in the dilution rate, D = 0.31 and
147
0.46 h-’ (Weber and San, 1989a). It should be noted that the behavior demonstrated by the par+ plasmid resembles that of the plasmid-containing culture exposed to random oscillations. However, the mixed population in the random cycling experiment is maintained at a higher plateau, reflecting a higher percentage, approximately 60 to 70%, of plasmid-containing cells. In addition, the plateau persisted only for approximately 100 h, half the time period of the plateau observed with the par+ plasmid. In the random cycling experiment the stability was believed to be caused by the random perturbations of the dilution rate. An environment is thus created which is more advantageous to the plasmid-containing cells. As the culture with the par-locus was not exposed to any imposed oscillations a different mechanism is responsible for the stability of the culture. Two possible theories are explored. One possibility is due to plasmid conjugation. Similar results were predicted by a model describing a conjugative plasmid system (Esty, 1986). However, this is probably not the case as the plasmid pBR322 is not conjugative. In addition, there was no evidence of conjugation by the par+ plasmid in subsequent experiments. The presence of the par-locus is a more probable explanation. If it is assumed that a decrease in the plasmid-containing population is accompanied by a decrease in the plasmid copy number (Weber and San, 1990) a population with a lower plasmid copy number exists during the stable plateau. The par-locus, in turn, might be more efficient in evenly dividing the plasmids between the daughter cells at a lower copy number. This could also result in the observed stable mixed populations. The final decrease in the percentage of plasmid-containing cells is most likely due to the ‘ take-over’ of the plasmid-free cells and not a failure of the par-locus. Restriction enzyme patterns of the plasmid pBR322-par isolated from the later part of the experiment do not show any major structural changes (data not shown). The advantage of the par-locus is an increase in the accuracy of plasmid partitioning between two daughter cells during cell division. Based on this assump-
300
200 Time
Fig. 3. Percentage 50 (0). 100 (0).
(h)
of plasmid-containing cells that formed colonies on plates supplemented with 25 (0). and 25 mg 1-l tetracycline (v) for plasmid 500 (A), and 2000 (A) mg 1-l ampicillin pBR322-par at D = 0.61 h-‘.
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showing the percentage of plasmid-containing cells that formed colonies between a supplemented plates for pBR322-par at D = 0.61 h-’ from 90.5 to 259.25 h. (b) the percentage of plasmid-containing cells that formed colonies between a range of supplemented plates for pBR322-par at D = 0.61 hh’ from 283 to 450 h.
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tion, the par+ culture should be able to completely stabilize the culture. However, as shown in Fig. 2a, the addition of the par-locus to a plasmid does not necessarily ensure full stability. In addition, as seen in Figs. 3 and 4, the culture composition is
150
200
300
400
500
Time (h) Fig.
5. Comparison
of the specific plasmid pBR322
p-lactamase activity, based on the total protein (0) and plasmid pBR322-par (W) at D = 0.61 h-‘.
present,
for
rhe
not homogeneous as expected with respect to the copy number, but heterogeneous, similar to the par- systems (Weber and San, 1990). Sub-populations, based on the MIC study, can be observed after 100 h. This corresponds to the transient period when the plasmid-free cells begin to emerge. The behavior of the population distribution is also similar to the previous experiments (Fig. 4 a,b). In the par+ culture, however, the sub-populations are more evenly distributed during the stable plateau. A gradual shift of one sub-population to another is also observed. It was suggested that the sub-population reflects a heterogeneity of the plasmid copy number (Weber and San, 1990). The stable period, 200 to 375 h, consists of a large percentage of low copy number cells. This would agree with one of the assumptions proposed to explain the increase in stability of the par+ culture. The par-locus has been shown to increase the stability of many plasmids (Lee and Edlin, 1985; Skogman et al., 1983; Zurita et al., 1984). However, there have been no reports of its effect on plasmid copy number and expression. A comparison of the P-lactamase activity of the par+ and par- plasmids is shown in Fig. 5. In the first 250 h there is no noticeable difference between the two cultures. After this time the activity of the par+ culture increases slightly then gradually decreases. This trend corresponds to the period, up to 375 h, when the plasmids are stably maintained. In general, it would appear that the addition of the par-locus, except for prolonging the period of productivity, has no significant effect on the plasmid expression. Shown in Fig. 6 is the relative plasmid copy number, defined as the plasmid DNA per chromosome DNA. It can be seen that although the behavior is similar between the pBR322 and pBR322-par cultures, the population containing the par-locus has a higher relative plasmid copy number. This could be caused by the effective partitioning of the plasmids in the par+ cells resulting in a decrease in the net rate of plasmid loss. The increase in the lag time, Fig. 2a, also supports this
151
‘0 Time
(h)
Fig. 6. Comparison of the relative plasmid copy number, plasmid DNA per chromosomeDNA, for plasmidpBR322(0) and plasmidpBR322-par(U) a1D = 0.61 h-‘. assumption. The rapid decrease, from 100 to 200 h, in the plasmid content may be due to the appearance of the plasmid-free cells and a gradual loss of plasmid per cell. In summary, the addition of the par-locus to the pBR322 plasmid under prolonged cultivation proved beneficial. Both the stability and the plasmid copy number of the culture increased. Although there was no immediate effect on the P-lactamase activity, the long-term productivity increased. However, until the mechanism of the par function is better understood, the exact relationship between stability, plasmid copy number, and expression and the par-locus is not clear.
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Hakkaart, M.J.J., Veltkamp, E. and Nijkamp, H.J.J. (1982a) Maintenance of the bacteriocinogenic plasmid Clo maintenance.
DF13 Mol.
in Escherichio coli cells, II. Specific Gen. Genet. 188, 338-344.
recombinant
functions
involved
in plasmid
Hakkaart, M.J.J., Wesseling,J.G., Veltkamp, E. and Nijkamp, H.J.J. (1982b) Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia co/i cells, 1. Localization and mutual interactions of four Clo DF13 incompatibility regions. Mol. Gen. Genet. 186, 531-539. Hinchliffe, E., Kuempel, P.L. and Masters, M. (1981) Escherichia co/i minichromosomes containing the pSClOI partitioning locus are not stably inherited. Plasmid 9, 286-297.
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Jacob, F., Brenner. S. and Cuzin, F. (1963) On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28, 329-348. in pBR322 derivatives reduces the Lee, S.W. and Edlin, G. (1985) Expression of tetracycline resistan reproductive fitness of plasmid-containing Escherichia coti. Gene3 9, 173-180. Meacock. P. and Cohen, S.N. (1980) Partitioning of bacterial plasmids during cell division: a Cis-acting locus that accomplishes stable plasmid inheritance. Cell 20, 529-542. Miki, T., Easton, A.M. and Rownd. R.H. (1980) Cloning of replication, incompatibility and stability functions of R plasmid NRl. J. Bacterial. 141. 87-99. Nordstrom. K., Molin. S. and Aagaard-Hansen, H. (1980) Partitioning of plasmid Rl in Esclrerichia co/i I. Kinetics of loss of plasmid derivative deleted of the par region. Plasmid 4, 215-227. Nordstrom, K. and Aagaard-Hansen, H. (1984a) Maintenance of bacterial plasmids: comparison of theoretical calculations and experiments with plasmid Rl in Escherichia co/i. Mol. Gen. Genet. 197, Nordstrom, K., Molin, S. and Light, J. (1984b) Control of replication of the bacterial plasmids: genetics. molecular biology and physiology of the plasmid Rl system. Plasmid 12, 71-90. Ogura, T. and Hiraga, S. (1983) Partition mechanism of plasmid: IWO plasmid gene-encoded products and a Cis-acting region are involved in partition. Cell 32, 351-360. Scott, J.R. (1984) Regulation of plasmid replication. Microbial. Rev. 48, l-23. Skogman, G., Nilsson, J. and Gustafsson, P. (1983) The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia co/i. Gene 23, 105. Weber, A.E. and San, K.-Y. (1988) Enhanced plasmid maintenance in a CSTR upon square-wave oscillations in the dilution rate. Biotechnol. Lett. 10. 531-536. Weber. A.E. and San K.-Y. (1989a) Dynamics of plasmid maintenance in a CSTR upon square-wave perturbations in the dilution rate, Biotechnol. Bioeng. 34, 1104-1113. Weber. A.E. and San, K.-Y. (1989b) A comparison of two plating techniques to estimate plasmid stability of a prolonged chemostat culture. Biotechnol. Tech. 3, 397-400. Weber, A.E. and San, K.-Y. (1990) Population dynamics of a recombinant culture in a chemostat under prolonged cultivation. Biotechnol. Bioeng. 36, 727-736. Zurita, M., Bolivar, F. and Sober6n, X. (1984) Construction and characterization of new cloning vehicles VII. Construction of plasmid pBR327par. a completely sequenced stable derivative of pBR327 containing the par locus of pSC101. Gene 28, 119-122.
