PLASMID 28, 70-79 (I 992)
Plasmid pTB913 Derivatives Are Segregationally Stable in Bacillus subtilis at Elevated Temperatures LINDA OSKAM, GERARD VENEMA, AND SIERD BRON' Department of Genetics, Centre of Biological Sciences, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands Received October 9, 199 1; revised February
18, 1992
We studied the effects of temperature on the segregational stability of derivatives of the rolling-circle-type plasmid pTB9 13 in Bacillus subtilis. This 4.5kb plasmid is a deletion derivative of pTB 19, which was originally isolated from a thermophilic Bacillus. pTB9 13 derivatives carrying large inserts or lacking the minus origin for complementary strand synthesis were segregationally unstable at 37°C. In contrast, at 47°C all pTB9 13 derivatives tested were stably maintained in B srtbtilis. The increased stability at 47°C was attributed, at least partly, to increased copy numbers at this temperature. Although considerable amounts of single-stranded and high-molecular-weight plasmid DNA were formed at 47°C these products did not reduce plasmid stability at this temperature. The increased stability and increased copy number of pTB9 I3 at clcvatcd tcmpcraturcs cxtcnd the USCofthis plasmid as a cloning vector in B. subtilis and other bacilli. 0 1992 Academic Press. Inc
used for cloning in gram-positive bacteria. In these organisms most of the plasmids studied seem to segregate randomly to daughter cells. As a consequence, the segregational stability of these plasmids will at least in part depend on plasmid copy numbers (for a review see Gruss and Ehrlich, 1989). Most plasmid vectors used in Bacillus replicate via single-stranded intermediates (ssDNA)~ by an asymmetric rolling-circle mechanism (RCR plasmids; te Riele et al., 1986; Gruss and Ehrlich, 1989). Evidence accumulated during the past few years suggests that the RCR mode of replication is an important factor in the high levels of instability generally observed with these plasmids (Bron, 1990; Bron et al., 199 1; Ehrlich et al., 1986; Gruss and Ehrlich, 1989). The insertion of foreign DNA into PUB 110 was shown to lead to a strong size-dependent decrease of the copy number of the circular plasmid forms and to increased levels of segregational
The stable maintenance of a plasmid in a bacterial cell is of prime importance for the development of efficient cloning vectors. However, instability has frequently been observed with recombinant plasmids in bacilli (for reviews see Bron, 1990; Ehrlich et al., 1986). Therefore, considerable attention has been directed to factors causing plasmid instability in bacilli, of which Bacillus subtilis has frequently been used as a model organism. Two types of plasmid instability are known: segregational instability, referring to the loss of the total plasmid population from the cell; and structural instability, referring to rearrangements-mostly deletions-in the plasmid DNA. Plasmid maintenance is governed by at least three factors: replication, differences in growth rates between plasmid-free and plasmid-containing cells, and partitioning. Partitioning is the process of distribution of plasmid copies to daughter cells during cell division. So far, no active partitioning functions have been described for plasmids commonly
’ To whom correspondence 0147-619X/92
* Abbreviations used: ssDNA, single-stranded DNA; RCR, replication; MO, minus origin; Km, kanamycin; Phleo, phleomycin.
should be addressed
$5.00
Capynght Cc?1992 by Acadcmlc Press. Inc. -\,I r@,,s ofrcproducuun ,,I any form reserved.
70
SEGREGATIONAL
PLASMID
instability (Bron and Luxen, 1985; Bron et al., 1988, 1991). Moreover, the instability of PUB 110 derivatives became more pronounced when the p&J type minus origin of replication (MO) was absent (Bron and Luxen, 1985; Bron et al., 1988). The positive effect of MOs on segregational plasmid stability seemsto be a general phenomenon, since it was also observed with pTA1060 (Bron et al., 1987, 1991) and pMV158 (W. J. J. Meijer, G. Venema, and S. Bron, unpublished results). The aim of the present studies was to test the maintenance of RCR plasmids at elevated temperatures. Such studies are relevant not only for the understanding of plasmid replication at high temperatures but also for the development of cloning vectors for thermophilic bacilli. pTB9 13, a 4.5-kb derivative of the 26.5-kb thermophilic Bacillus plasmid pTB 19 (Imanaka et al., 1984) was chosen for our purpose. It was shown previously that the nucleotide sequence and genetic organization of pTB913 were highly similar to those of the well-known RCR plasmid pUBll0 (Matsumura et al., 1984; Muller et al., 1986; van der Lelie et al., 1989). The results described here indicated that pTB913 derivatives were stably maintained at 47°C in B. subtihs and that this plasmid has a good potential asa cloning vector at elevated temperatures in bacilli. MATERIALS
AND METHODS
Bacterial Strains, Plasmids, and Media The strains and plasmids used in this study are listed in Table 1. LB medium used for culturing B. subtilis was described before (Maniatis et al., 1982); solid media contained 1.5% agar. Kanamycin (Km) and phleomytin (Phleo) were used at final concentrations of 8 and 1 pg/ml.
STABILITY
IN 8
\uhli/i\
71
(Mannheim, Germany) and used as recommended by the supplier. Molecular cloning procedures were performed as described by Maniatis et al. (1982). Enzyme-treated DNA was analyzed in horizontal 0.6% agarosegels in TBE buffer (89 mtvt Tris-borate, 89 mM boric acid, 2 mM EDTA, pH 8.0) which contained 0.5 pg ethidium bromide per milliliter. DNA fragments were isolated from gels using the freeze-squeeze method (Tautz and Renz, 1983). Isolation of Plasmid DNA and Transformation of B. subtilis Plasmid DNA was isolated following the alkaline-lysis procedures for Bacillus (Bron, 1990). Total DNA lysates were prepared as described by Bron (1990). B. subtilis 8G5 competent cells were prepared and transformed as described by Bron and Luxen (1985).
Southern Blot Hybridizations of Total DNA Lysates After electrophoresis of samples of total DNA lysates, each containing approximately 10 pg of DNA (chromosomal plus plasmid DNA) in 0.6% agarosegels containing 0.5 pg/ ml ethidium bromide, the DNA was transferred to GeneScreen Plus filters (NEN Research Products, Boston, MA) by Southern blotting as described by Chomczynski and Qasba (1984). Probe labeling, DNA hybridization conditions, and washing steps were performed using the ECL gene detection system (Amersham International plc, Amersham, UK). The procedures recommended by the manufacturers were followed. Plasmid Copy Number Determination
Plasmid copy numbers were determined basically as described by Bron ( 1990). Cells Restriction Enzyme Reactions, DNA were grown in LB medium which contained Manipulations, Molecular Cloning, and 50 &i/ml [methyl-3H]thymidine during the Gel Electrophoresis whole period of cultivation. Labeled plasmid Restriction enzymes and endonuclease S1 pluschromosomal DNA extracted from expowere purchased from Boehringer GmbH nentially growing cells were separated in aga-
72
OSKAM, VENEMA, AND BRON TABLE 1 BACTERIALSTRAINSAND PLASMIDS
Bacterial strain or plasmid Bacteria B. subtilis 168 8G5 Plasmids pTB19 pTB913 pTB913-1C pTB9 13-3C pTB3 pTB3-1C pTB3-3C
Properties
Source of reference
trpC2, tyrI, met, his, nit, purA, ura, rib
Bron and Venema (I 972)
KmR, Phleo’, TcR, 26.5 kb, derived from a thermophihc Bacillus RepB, KmR, PhleoR, 4.5-kb derivative of pTB 19 RepB, PhleoR, 5.8 kb, pTB9 13 containing a I .3kb E. coli DNA fragment, denoted 1C RepB, PhleoR, 8.7 kb, pTB913 containing a 4.2kb E. coli DNA fragment, denoted 3C RepB, KmR, PhleoR, ApaIU, 4.3 kb RepB, PhleoR, 5.6 kb, pTB3 containing the I .3kb 1C DNA fragment RepB, PhleoR, 8.5 kb, pTB3 containing the 4.2kb 3C DNA fragment
Imanaka et al. (1981)
rose gels and the radioactivities in the plasmid fraction (covalently closed plus open circular monomers) and chromosomal DNA fraction were used to estimate the copy numbers per chromosome equivalent.
Imanaka et al. ( 1984) This work This work van der Lelie et al. (1989) This work This work
nies showed that the resistance to Phleo was in all casesassociatedwith the presence of the plasmid. Likewise, sensitivity to Phleo was associated with the absence of the plasmid (50 colonies were tested). RESULTS
Segregational Plasmid Stability Assays
pT3913 Derivatives We studied the effects of three variables on Assays of segregational plasmid stabilities were performed essentially as described by the maintenance of plasmids derived from Bron and Luxen (1985). Precultures in LB pTB9 13: plasmid size, presence/absence of medium containing the selective antibiotic the palU MO, and temperature. The MO of (Phleo, 1 pg/ml) were grown at the test tem- pTB9 13 is identical to the MO of PUB 110 perature, In the late logarithmic growth (van der Lelie et al., 1989). The removal of phase the cells were diluted 104-fold into 25 the MO from pTB913 as a 226-bp FnuDIIDraI fragment, resulting in pTB3, was deml of nonselective medium. Approximately 1000 to 5000 cells were transferred to 25-ml scribed before (van der Lelie et al., 1989). To portions of fresh nonselective medium at 8-h vary the size of the plasmid, we cloned the 1C intervals, before the cells reached the station- (1.3 kb) and 3C (4.2 kb) DNA fragments, which had been randomly isolated from the ary phase. Samples taken from each dilution step were plated on nonselective plates to es- Escherichia coli chromosome (Bron and timate the number of generations that the Luxen, 1985) into the unique BglII site of cells had grown under nonselective condi- pTB9 13 and pTB3. This resulted in the plaskb), tions. Subsequently, at least 50 colonies were midspTB913-lC(5.8kb),pTB3-lC(5.6 individually transferred to selective plates to pTB913-3C (8.7 kb), and pTB3-3C (8.5 kb), estimate the percentage of cells that were re- all of which were introduced into B. subtilis sistant to Phleo. Plasmid analysis of 50 colo- 8G5 competent cells.
SEGREGATIONAL
PLASMID STABILITY
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FIG. 1. Segregational stabilities of pTB9 13 (A and C) and pTB3 derivatives (B and D) in B. subtilis strain 8G5. Stability assayswere performed in the absence of selective antibiotics at 37°C (A and B) or 47°C (C and D). Stabilities are expressed as the percentage of colonies resistant to the selective antibiotic (Phleo).
SegregationalStabilities of pTB913 Derivatives The segregational stabilities of the pTB9 13 and pTB3 series of plasmids were studied at 37 and 47°C. At 37°C pTB913 was stably maintained. A slight degree of plasmid loss was observed, however, upon insertion of the 1C and 3C DNA fragments (Fig. IA). In the absence of the MO, plasmid maintenance was considerably reduced with the largest plasmid, pTB3-3C (Fig. 1B). These results showed that at 37°C both the increase in plasmid size and the absence of the MO resulted in increased levels of plasmid instability. The plasmid stability assays at 47°C (Figs. 1C and 1D) gave a striking result: in contrast to the results obtained at 37°C all pTB9 13 derivatives, including those lacking the MO, were stably maintained at this temperature. As one possible cause of the high plasmid stability at 47°C we conceived that growth advantages of plasmid-free over plasmid-
containing cells, which might exist at 37°C would be absent or strongly reduced at 47°C. To study this possibility, temperature-shift experiments were performed. Two B. subtilis 8G5 (pTB3-3C) cultures were grown for about 60 generations, one at 37°C and the other at 47°C. Both cultures were then divided into two equal parts, one of which was shifted to 47°C and the other kept at 37°C. The fractions of plasmid-containing cells were determined at regular intervals after further incubation. The results of the stability assaysshowed that after the shift of the culture that had been preincubated at 37’C, plasmid-free cells accumulated with almost similar kinetics at 37 and 47°C (Fig. 2A). These results can be explained by growth advantage of plasmid-free over plasmid-containing cells at both 37 and 47°C (about 15% of the cells in the culture preincubated at 37°C were plasmid-free at the time of the shift). Support for this explanation was obtained in the following way. B. subtilis 8G-5 cells containing pTB3-3C were mixed with
74
OSRAM, VENEMA, AND BRON lOOf
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FIG. 2. The effect oftemperature shifts on the maintenance of pTB3-3C in B. subtilis. Cells were cultured for about 60 generations at 37 or 47°C under nonselective conditions, after which the cultures were separated in two equal parts. One part was maintained at 37°C and the other at 47°C. (A) Culture preincubated at 37°C; (B) culture preincubated at 47°C. Arrows indicate the temperature shift. (C) Cells containing pTB3-3C were mixed with plasmid-free cells. Overgrowth of plasmid-free cells was monitored at 37 and 47°C.
plasmid-free cells in a ratio of about 85:15. The mixed cultures were subsequently grown at either 37 or 47°C. The results of stability assaysof these mixed cell populations (Fig. 2C) confirmed that at both 37 and 47°C plasmid-free cells rapidly overgrew cells containing the plasmid. This result strongly supports the view that in the experiment shown in Fig. 2A the overgrowth of plasmid-free cells caused their accumulation at 37 and 47°C. Since the overgrowth by plasmid-free cells was even slightly faster at 47 than at 37”C, the observed stabilization of pTB9 13 derivatives at 47°C did not, apparently result from reduced differences in growth rates between plasmid-free and plasmid-containing cells at this temperature. The results obtained with the culture that was preincubated at 47°C are shown in Fig. 2B. In this culture, plasmid-free cells were neither present at the time of the shift nor
formed after continued incubation at 47°C. This confirmed the conclusion from Figs. 1C and 1D that pTB9 13 derivatives were stably maintained at 47°C. After the temperature of this culture was shifted to 37 “C, plasmid-free cells accumulated rapidly, however. Apparently, the preincubation of the cells at 47°C could not prevent the subsequent accumulation of plasmid-free cells at 37°C. Together, the results presented in this section indicate that the primary cause of pTB913 stabilization at 47°C was a lowering in the probability of segregating plasmid-free cells. Once such cells were formed, they appeared to outcompete plasmid-containing cells rapidly, however, at both 37 and 47°C. Plasmid ProJles and Copy Numbers
Plasmids using RCR generate ssDNA (te Riele et al., 1986; Gruss and Ehrlich, 1989)
SEGREGATIONAL * -+-+-
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PLASMID STABILITY
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75
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FIG. 3. Plasmid patterns in total DNA lysates from B. .sz~h/ili.s XC5 harboring pTB9 I3 or pTB3 derivatives. (+) and (-) indicate that the DNA preparations were incubated with endonuclease Sl or left untreated. Left panel, 37°C; right panel. 47°C. (A) pTB9 13: (B) pTB9 I3- 1C; (C) pTB9 I3-3C: (D) pTB3; (E) pTB3-1C; and (F): pTB3-3C. The positions of ssDNA (>) and HMW DNA are indicated.
and, under certain conditions, HMW DNA in B. subtilis (Gruss and Ehrlich, 1988, 1989; Viret and Alonso, 1987; Viret et al., 1991). To investigate whether such replication products were also formed in B. subtilis under the different temperature conditions used in the present studies, total DNA lysates extracted from the various plasmid-containing cells were analyzed using Southern hybridizations. The results (Fig. 3) showed that ssDNA and HMW DNA were indeed present in certain plasmid preparations. However, no large differences were observed between the plasmid profiles from cultures grown at 37 or 47°C. For instance, significant amounts of ssDNA were found in the absenceof the MO, in particular with the smaller plasmids (Fig. 3, lanes D), but the relative amounts of ssDNA were roughly similar at 37 and 47°C. Also, small amounts of plasmid HMW DNA were present with plasmids carrying the 1C fragment (Fig. 3, lanes B), but the relative amounts of this DNA were about the same at 37 and 47°C. It proved difficult to produce clear hybridization profiles of the plasmids carrying the 3C insert: aspecific degradation of the plasmid DNA was consistly observed. Despite this difficulty, it was clear that with these plasmids large amounts of plasmid DNA were present at the HMW position (Fig. 3, lanes C and F) and that, again, there
were no striking differences between the results obtained at 37 and 47°C. Basedon these results, we concluded that the temperature (37 or 47°C) had no drastic effect on the amounts of ssDNA and HMW DNA relative to the other plasmid forms. Therefore, it is unlikely that gross alterations in the production of ssDNA and HMW plasmid DNA caused the high stability of pTB913 derivatives at 47°C. We next studied whether the temperature affected the copy numbers of the circular plasmid monomers (the amounts of circular multimeric molecules were low). Although it was difficult to reproduce the copy number estimations quantitatively, we were able to determine the relative differences in copy numbers in a reproducible manner (three independent experiments were carried out). The averaged relative copy numbers per chromosome equivalent (Fig. 4) appeared to be reduced by either the insert (lC, about twofold; 3C, about fourfold) or by the absence of the MO (about twofold). In contrast, the relative copy numbers per chromosome equivalent were increased by a factor of about two when the temperature was raised from 37 to 47°C. In the method used here for the copy number determinations per chromosome equivalent, the eventual presence of linear HMW plasmid DNA, which migrates
76
OSKAM, VENEMA, AND BRON
;miii
pTE313 plTB13-1c pTE!P13-x plP3 t%sm!d
pTB3-1c
pm3-3c
FIG. 4. Relative plasmid copy numbers in B. subtilis 8G5 at 37 and 47°C. Copy numbers of the monomeric circular plasmid forms were determined per chromosome equivalent. The entries in the figure, presented as relative copy numbers, were the average ofthree independent experiments. As a standard, the copy number of pTB9 I3 at 37°C was set at 100%:this represents an actual copy number of approximately 30 per chromosome equivalent.
DNA inserts and the removal of the MO resulted in reductions of the plasmid copy numbers. Random partitioning of the available plasmid copies would then generate plasmidfree cells at a higher frequency. In agreement with this explanation was the observation in the present work that increased plasmid size or deletion of the MO resulted in reduced copy numbers. We can conceive of at least four possible causesfor the high segregational stability observed with pTB913 derivatives at 47°C: (i) effects of a heat-shock response, (ii) effectson ssDNA and HMW plasmid DNA formation; (iii) reduced differences in growth rates between plasmid-free and plasmid-containing cells, and (iv) increased copy numbers. These possibilities are discussed in the following sections. (i) Effectsof a Heat-Shock Response
together with the chromosal DNA fraction (Gruss and Ehrlich, 1988) might result in underestimates of the plasmid copy numbers. Since we estimated that the amounts of HMW plasmid DNA observed in the present studies were at most 10% of the chromosomal DNA (with plasmid pTB913-3C), we did not correct for possible errors due to HMW plasmid DNA.
Although no experimental evidence is available, one might speculate that in some unknown way the induction of heat-shock proteins was involved in the plasmid stabilization in B. subtilis at 47°C. In B. subtilis an impressive heat-shock response, involving as many as 66 proteins, has been observed (Miller et al., 1991). Moreover, it is known that some heat-shock-induced genes are involved in the replication of E. coli plasmids (Tilly and Yarmolinski, 1989; Kawasaki et DISCUSSION al., 1990). A role of heat-shock proteins in In the present work we studied the effects plasmid replication and plasmid stability in of the growth temperature of B. subtilis on B. subtilis remains to be established, howthe maintenance of pTB913, an RCR plasever. mid originating from a thermophilic Bacillus. The most striking result was that all (ii) Eflhcts on ssDNA and HMW Plasmid pTB9 13 derivatives tested were stably mainDNA Formation tained at 47°C although several derivatives Based on several studies it has been sugwere segregationally unstable at 37°C. Two parameters appeared to reduce the gested that the accumulation of ssDNA maintenance of pTB9 13 derivatives at 37°C: (Bron et al., 1985, 1988, 1991; Gruss et al., (i) the absence of a functional MO, and (ii) 1987; Viret and Alonso, 1987) or HMW increased plasmid size. These results are remi- DNA (Gruss and Ehrlich, 1988, 1989; Viret niscent of those described previously for et al., 1991) may reduce plasmid maintepUBll0 (Bron and Luxen, 1985; Bron et al., nance. The lower growth rates of plasmid1988, 1991). One possible explanation for containing compared to those of plasmidthese observations is that both the cloning of free cells, resulting in the overgrowth of the
SEGREGATlONAL
PLASMID
latter, have been considered as a likely cause of the reduced plasmid stability. In the present studies we showed that at 37°C reduced plasmid maintenance was associatedwith increased amounts of ssDNA (absence of the MO) or HMW DNA (presence of DNA inserts). This leaves the possibility open that at 37°C these replication products contributed to plasmid instability. At 47”C, the relative amounts of ssDNA and HMW DNA formed by pTB913 derivatives were, however, roughly similar to those at 37°C. This means that the plasmid stabilization observed at 47°C was not the consequence of a reduced accumulation of ssDNA or HMW DNA. Clearly, the considerable amounts of ssDNA and HMW DNA that accumulated at 47°C with some pTB9 13 derivatives did not reduce plasmid maintenance at this temperature. This could mean that the general idea that ssDNA/HMW DNA interfere with plasmid maintenance is incorrect or, alternatively, that specifically at the elevated temperature the harmful effects of these replication products are reduced. If the latter possibility is true, then one could speculate that the heatshock response described in the foregoing section plays a role in this process.
STABILITY
IN L1. .mh/i/i\
17
(iv) Increased Copy Numbers
We consider this the most attractive explanation for at least part of the observed plasmid stabilization effects at 47°C. The pTB913 copy numbers per chromosome equivalent were approximately twofold higher at 47 than at 37°C. This corroborates results by Shivakumar and Dubnau ( 1978), which indicated that at 47°C the amount of pUBll0 DNA was increased about twofold in B. subtilis. The increased copy numbers are expected to reduce the probability of generating plasmid-free cells. Copy number levels cannot, however, explain all observations in the present studies. For instance, plasmid pTB9 13- 1C showed a low, yet distinct degree of instability at 37”C, while plasmid pTB33C was fully stable at 47°C. Nevertheless, the copy number of pTB9 13-1C at 37°C was higher than that of pTB3-3C at 47°C. The nature of the putative additional factor increasing the stability of pTB9 13 plasmids at 47°C is not clear. As already discussed, we consider the heat-shock response a possible candidate for this function. The cause of the increased copy numbers at 47°C is a matter of speculation. Apparently, the copy control system enables the establishment of higher plasmid levels at 47°C. (iii) Reduced Diferences in Growth Rates Several explanations can be entertained for betweenPlasmid-Free and Plasmidthis. One is that at the higher temperature the Containing Cells interaction between the replication origin Growth advantage of plasmid-free over and the rate-limiting replication initiation plasmid-containing cells can be an important protein is more efficient, This might, for infactor in plasmid instability in bacteria (Bee stance, be due to altered sup&&city of the et al., 1987). This also appeared to be the case plasmid DNA at 47°C. Gennaro and Novick in the studies with pTB9 13 reported here, (I!%@), who analyzed the RCR plasmid Once formed, plasmid-free c&s appeared to pT 181, have indicated that the interaction beoutcompete plasmid-containing cells rapidly, tween the origin and the replication protein is at both 37 and 47°C. Reduced di&rew in imp&ant for rapid readjustments of the growth rates between plasm&free and p& copy number after, for instance, cell division. mid-containing cells cannot, therefore, ex- In out ,,iiem, a more efficient interaction plain the observed plasmid stabilization at between the pTB913 origin and the replica47°C. Our results rather indicated that the tion protein at 47°C might enable more stabilization of pTB9 13 derivatives at 47Y rounds of plasmid replication during the was due primarily to events that reduced the short life cycle of the cell, which lasts for only probability of segregating plasmid-free cells about 20 min at 47°C. The second possible from plasmid-containing parents. explanation is that more replication initia-
78
OSKAM,
VENEMA,
tion protein is produced at the higher temperature. This could be mediated through an altered efficiency of interaction of pTB9 13 antisense RNAs with the mRNA specifying the replication initiation protein. It has been shown that antisense RNA is important in the regulation of the copy number of plasmid pT 181 (Kumar and Novick, 1985). For pLS1 (Perez-Martinetal., 1988)andpUBllO(Maciag et al., 1988) which is very similar to pTB9 13, regulation of copy numbers via antisense RNA has been proposed. This makes it attractive to believe that antisense RNA also plays a role in the regulation of pTB9 13 copy numbers. In conclusion, these studies showed that in B. subtilis the stable maintenance of pTB9 13 derivatives can be realized by growing the cell cultures at elevated temperatures. This is a valuable property for the development of this plasmid as a cloning vector for bacilli. We have already extended our studies to thermophilic bacilli: in certain strains of Bacillus stearothevmophilus,pTB9 13 derivatives appeared to be fairly stable up to at least 57°C (unpublished results). So far, we have not tested whether RCR plasmids other than pTB913 are stably maintained at elevated temperatures, but, given the high degree of relatedness between various RCR plasmids (Gruss and Ehrlich, 1989) we consider this a likely possibility. ACKNOWLEDGMENTS This work was supported by the Program Commission Industrial Biotechnology of the Ministry of Economic Affairs (The Netherlands). We thank Jan Maarten van Dijl for valuable discussions, Arie Hamminga for preparation of the media, and Henk Mulder for preparing the figures.
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(I 987). Stability function in Bacillus subtilis plasmid pTAl060. Plasmid l&8-15. BRON, S., HAIMA, P., MEIJER, W., OSKAM, L., HOLSAPPEL, S., AND VENEMA, G. (I 99 1). Plasmid (in)stability in Bacillus subtilis. In “Proceedings ofthe 6th International Symposium on Genetics of Industrial Microorganisms” (H. Heslot, J. Davies, J. Florent, L. Bobichon, G. Durand, and L. Penasse, Eds.). Vol. I, pp. 49-60. SociettC Francaise de Microbiologic, Strasbourg, France. BRON, S., AND LUXEN, E. (1985). Segregational instability ofpUB IO-derived recombinant plasmids in Bacil-
lus subtilis. Plasmid 14, 235-244. BRON, S., LUXEN, E., AND SWART, P. (1988). Instability of recombinant pUBl IO plasmids in BuciNus subtilis. plasmid-encoded stability function and effects of DNA inserts. Plasmid 19,23 l-241. BRON, S., AND VENEMA, G. (1972). Ultraviolet inactivation and excision repair in BaciNus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat. Res. 15, l-10. CHOMCZYNSIU, P., AND QASBA, P. K. (I 984). Alkaline transfer of DNA to plastic membrane. Biochem.
Biophys. Res. Common. 122, 340-344. EHRLICH, S. D., NOIROT, P., PETIT, M. A., JANN~RE, L., MICHEL, B., AND TE RIELE, H. (I 986). Structural instability of Bacillus subtilis plasmids. In “Genetic Engineering” (J. K. Setlow, and A. Hollaender, Eds.), Vol. 8., pp. 71-83. Plenum, New York. GENNARO, M. L., AND NOVICK. R. P. (1988). An enhancer of DNA replication. J. Bacterial. 170, 57095717. GRUSS, A. D., AND EHRLICH, S. D. ( 1988). Insertion of foreign DNA into plasmids from gram-positive bacteria induces formation of high-molecular-weight plasmid multimers. J. Bacterial. 170, I I83- I 190. GRUSS, A. D., AND EHRLICH, S. D. (1989). The family of highly interrelated ssDNA plasmids. Microbial. Rev. 53,231-241. GRUSS, A. D., Ross, H. F., AND NOVICK, R. P. (I 987). Functional analysis of a palindromic sequence required for normal plasmid replication ofseveral staphylococcal plasmids. Proc. Natl. Acad. Sci. USA 84, 2165-2169. IMANAKA, T., ANO, T., FUJII, M., AND AIBA, S. (1984). Two replication determinants of an antibiotic-resistance plasmid, pTB 19, from a thermophilic Bacillus. J. Gen. Microbial. 130, 1399-1408. IMANAKA, T., FUJII, M., AND AIBA, S. (1981). Isolation and characterization of antibiotic resistance plasmids from thermophilic Bacilli and construction of deletion plasmids. J. Bacterial. 146, 109 I - 1097. KAWASAKI, Y., WADA, C., AND YURA, T. (1990). Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication. Mol. Cm.
Genet. 220,277-282. KUMAR, C. C., AND NOVICK, R. P. (1985). Plasmid
SEGREGATIONAL
PLASMID STABILITY
pT18 I replication is regulated by two countertranscripts. Proc. Nut,. Acud. Sci. I!SA 82, 638-642. MACIAG, I. E., VIRET, J.-F., AND ALONSO,J. C. (1988). Replication and incompatibility properties of plasmid pUB1 IO in Buciiius snbtilis. Mol. Gen. Genet. 212, 232-240. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK.J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MATSUMURA,M., KATAKURA, Y., IMANAKA, T., AND AIBA, S. (I 984). Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUBl10. J. Bacterial. 160,4 13-420.
MILLER, B. S., KENNEDY, T. E., AND STREIPS,U. N. (199 I). Molecular characterization of specific heat shock proteins in Bacillus subtilis. Current Microbial. 22,23 l-236. MULLER, R. E., ANO, T., IMANAKA, T., AND AIBA, S. (1986). Complete nucleotide sequences of Bacillus plasmids pUB1 lOdB, pRBH1 and its copy mutants. Mol. Gen. Genet. 202, 169-111. PEREZ-MARTIN,J., DEL SOUR, G. H., DE LA CAMPA, A. G., AND ESPINOSA,M. (1988). Three regions in the
IN 1y. slrhrilr~
79
DNA of plasmid pLSl show sequence-directed static bending. n;uc,leic,-lcidc REY.16, 9 113-9 126. SHIVAKUMAR,A. G.. AND DUBNAU. D. (1978). Plasmid replication in &I Ts mutants of Bacillus rubtrlrc Plusmid 1, 405-416. TAUTZ, D.. AND RENZ, M. (1983). An optimized freezesqueeze method for the recovery of DNA fragments from agarose gels. Anal. Biochem. 132, 14- 19. TE RIELE, H., MICHEL. B., AND EHRLICH,S. D. ( 1986). Single-stranded plasmid DNA in BaciNus snbtilis and Stuphylococcusaweus. Proc. Natl. Acud. Sci. 1:X4 83, 2541-2545. TILLY. K., AND YARMOLINSKI,M. (1989). Participation of Escherichia coli heat shock proteins DnaJ. DnaK and GrpE in PI plasmid replication. J. Bacterial. 171, 6025-6029. VAN DERLELIE,D., BRON,S., VENEMA,G., AND OSKAM, L. (1989). Similarity of minus origins and flanking open reading frames of plasmids PUB I IO, pTB9 13 and pMV 158. Nucleic Acids Res. 17, 7283-7294. VIRET, J.-F., AND ALONSO,J. C. (1987). Generation of multigenome-length plasmid molecules in BaciNus subtilis. Nucleic .4cidJ Rex 15, 6349-6368. VIRET, J.-F., BRAVO,A., AND ALONSO,J. C. (199 I). Recombination-dependentconcatemericplasmidreplication. Microbial. Rev. 55, 675-683. Communicated bJ
Plasmid pTB913 Derivatives Are Segregationally Stable in Bacillus subtilis at Elevated Temperatures LINDA OSKAM, GERARD VENEMA, AND SIERD BRON' Department of Genetics, Centre of Biological Sciences, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands Received October 9, 199 1; revised February
18, 1992
We studied the effects of temperature on the segregational stability of derivatives of the rolling-circle-type plasmid pTB9 13 in Bacillus subtilis. This 4.5kb plasmid is a deletion derivative of pTB 19, which was originally isolated from a thermophilic Bacillus. pTB9 13 derivatives carrying large inserts or lacking the minus origin for complementary strand synthesis were segregationally unstable at 37°C. In contrast, at 47°C all pTB9 13 derivatives tested were stably maintained in B srtbtilis. The increased stability at 47°C was attributed, at least partly, to increased copy numbers at this temperature. Although considerable amounts of single-stranded and high-molecular-weight plasmid DNA were formed at 47°C these products did not reduce plasmid stability at this temperature. The increased stability and increased copy number of pTB9 I3 at clcvatcd tcmpcraturcs cxtcnd the USCofthis plasmid as a cloning vector in B. subtilis and other bacilli. 0 1992 Academic Press. Inc
used for cloning in gram-positive bacteria. In these organisms most of the plasmids studied seem to segregate randomly to daughter cells. As a consequence, the segregational stability of these plasmids will at least in part depend on plasmid copy numbers (for a review see Gruss and Ehrlich, 1989). Most plasmid vectors used in Bacillus replicate via single-stranded intermediates (ssDNA)~ by an asymmetric rolling-circle mechanism (RCR plasmids; te Riele et al., 1986; Gruss and Ehrlich, 1989). Evidence accumulated during the past few years suggests that the RCR mode of replication is an important factor in the high levels of instability generally observed with these plasmids (Bron, 1990; Bron et al., 199 1; Ehrlich et al., 1986; Gruss and Ehrlich, 1989). The insertion of foreign DNA into PUB 110 was shown to lead to a strong size-dependent decrease of the copy number of the circular plasmid forms and to increased levels of segregational
The stable maintenance of a plasmid in a bacterial cell is of prime importance for the development of efficient cloning vectors. However, instability has frequently been observed with recombinant plasmids in bacilli (for reviews see Bron, 1990; Ehrlich et al., 1986). Therefore, considerable attention has been directed to factors causing plasmid instability in bacilli, of which Bacillus subtilis has frequently been used as a model organism. Two types of plasmid instability are known: segregational instability, referring to the loss of the total plasmid population from the cell; and structural instability, referring to rearrangements-mostly deletions-in the plasmid DNA. Plasmid maintenance is governed by at least three factors: replication, differences in growth rates between plasmid-free and plasmid-containing cells, and partitioning. Partitioning is the process of distribution of plasmid copies to daughter cells during cell division. So far, no active partitioning functions have been described for plasmids commonly
’ To whom correspondence 0147-619X/92
* Abbreviations used: ssDNA, single-stranded DNA; RCR, replication; MO, minus origin; Km, kanamycin; Phleo, phleomycin.
should be addressed
$5.00
Capynght Cc?1992 by Acadcmlc Press. Inc. -\,I r@,,s ofrcproducuun ,,I any form reserved.
70
SEGREGATIONAL
PLASMID
instability (Bron and Luxen, 1985; Bron et al., 1988, 1991). Moreover, the instability of PUB 110 derivatives became more pronounced when the p&J type minus origin of replication (MO) was absent (Bron and Luxen, 1985; Bron et al., 1988). The positive effect of MOs on segregational plasmid stability seemsto be a general phenomenon, since it was also observed with pTA1060 (Bron et al., 1987, 1991) and pMV158 (W. J. J. Meijer, G. Venema, and S. Bron, unpublished results). The aim of the present studies was to test the maintenance of RCR plasmids at elevated temperatures. Such studies are relevant not only for the understanding of plasmid replication at high temperatures but also for the development of cloning vectors for thermophilic bacilli. pTB9 13, a 4.5-kb derivative of the 26.5-kb thermophilic Bacillus plasmid pTB 19 (Imanaka et al., 1984) was chosen for our purpose. It was shown previously that the nucleotide sequence and genetic organization of pTB913 were highly similar to those of the well-known RCR plasmid pUBll0 (Matsumura et al., 1984; Muller et al., 1986; van der Lelie et al., 1989). The results described here indicated that pTB913 derivatives were stably maintained at 47°C in B. subtihs and that this plasmid has a good potential asa cloning vector at elevated temperatures in bacilli. MATERIALS
AND METHODS
Bacterial Strains, Plasmids, and Media The strains and plasmids used in this study are listed in Table 1. LB medium used for culturing B. subtilis was described before (Maniatis et al., 1982); solid media contained 1.5% agar. Kanamycin (Km) and phleomytin (Phleo) were used at final concentrations of 8 and 1 pg/ml.
STABILITY
IN 8
\uhli/i\
71
(Mannheim, Germany) and used as recommended by the supplier. Molecular cloning procedures were performed as described by Maniatis et al. (1982). Enzyme-treated DNA was analyzed in horizontal 0.6% agarosegels in TBE buffer (89 mtvt Tris-borate, 89 mM boric acid, 2 mM EDTA, pH 8.0) which contained 0.5 pg ethidium bromide per milliliter. DNA fragments were isolated from gels using the freeze-squeeze method (Tautz and Renz, 1983). Isolation of Plasmid DNA and Transformation of B. subtilis Plasmid DNA was isolated following the alkaline-lysis procedures for Bacillus (Bron, 1990). Total DNA lysates were prepared as described by Bron (1990). B. subtilis 8G5 competent cells were prepared and transformed as described by Bron and Luxen (1985).
Southern Blot Hybridizations of Total DNA Lysates After electrophoresis of samples of total DNA lysates, each containing approximately 10 pg of DNA (chromosomal plus plasmid DNA) in 0.6% agarosegels containing 0.5 pg/ ml ethidium bromide, the DNA was transferred to GeneScreen Plus filters (NEN Research Products, Boston, MA) by Southern blotting as described by Chomczynski and Qasba (1984). Probe labeling, DNA hybridization conditions, and washing steps were performed using the ECL gene detection system (Amersham International plc, Amersham, UK). The procedures recommended by the manufacturers were followed. Plasmid Copy Number Determination
Plasmid copy numbers were determined basically as described by Bron ( 1990). Cells Restriction Enzyme Reactions, DNA were grown in LB medium which contained Manipulations, Molecular Cloning, and 50 &i/ml [methyl-3H]thymidine during the Gel Electrophoresis whole period of cultivation. Labeled plasmid Restriction enzymes and endonuclease S1 pluschromosomal DNA extracted from expowere purchased from Boehringer GmbH nentially growing cells were separated in aga-
72
OSKAM, VENEMA, AND BRON TABLE 1 BACTERIALSTRAINSAND PLASMIDS
Bacterial strain or plasmid Bacteria B. subtilis 168 8G5 Plasmids pTB19 pTB913 pTB913-1C pTB9 13-3C pTB3 pTB3-1C pTB3-3C
Properties
Source of reference
trpC2, tyrI, met, his, nit, purA, ura, rib
Bron and Venema (I 972)
KmR, Phleo’, TcR, 26.5 kb, derived from a thermophihc Bacillus RepB, KmR, PhleoR, 4.5-kb derivative of pTB 19 RepB, PhleoR, 5.8 kb, pTB9 13 containing a I .3kb E. coli DNA fragment, denoted 1C RepB, PhleoR, 8.7 kb, pTB913 containing a 4.2kb E. coli DNA fragment, denoted 3C RepB, KmR, PhleoR, ApaIU, 4.3 kb RepB, PhleoR, 5.6 kb, pTB3 containing the I .3kb 1C DNA fragment RepB, PhleoR, 8.5 kb, pTB3 containing the 4.2kb 3C DNA fragment
Imanaka et al. (1981)
rose gels and the radioactivities in the plasmid fraction (covalently closed plus open circular monomers) and chromosomal DNA fraction were used to estimate the copy numbers per chromosome equivalent.
Imanaka et al. ( 1984) This work This work van der Lelie et al. (1989) This work This work
nies showed that the resistance to Phleo was in all casesassociatedwith the presence of the plasmid. Likewise, sensitivity to Phleo was associated with the absence of the plasmid (50 colonies were tested). RESULTS
Segregational Plasmid Stability Assays
pT3913 Derivatives We studied the effects of three variables on Assays of segregational plasmid stabilities were performed essentially as described by the maintenance of plasmids derived from Bron and Luxen (1985). Precultures in LB pTB9 13: plasmid size, presence/absence of medium containing the selective antibiotic the palU MO, and temperature. The MO of (Phleo, 1 pg/ml) were grown at the test tem- pTB9 13 is identical to the MO of PUB 110 perature, In the late logarithmic growth (van der Lelie et al., 1989). The removal of phase the cells were diluted 104-fold into 25 the MO from pTB913 as a 226-bp FnuDIIDraI fragment, resulting in pTB3, was deml of nonselective medium. Approximately 1000 to 5000 cells were transferred to 25-ml scribed before (van der Lelie et al., 1989). To portions of fresh nonselective medium at 8-h vary the size of the plasmid, we cloned the 1C intervals, before the cells reached the station- (1.3 kb) and 3C (4.2 kb) DNA fragments, which had been randomly isolated from the ary phase. Samples taken from each dilution step were plated on nonselective plates to es- Escherichia coli chromosome (Bron and timate the number of generations that the Luxen, 1985) into the unique BglII site of cells had grown under nonselective condi- pTB9 13 and pTB3. This resulted in the plaskb), tions. Subsequently, at least 50 colonies were midspTB913-lC(5.8kb),pTB3-lC(5.6 individually transferred to selective plates to pTB913-3C (8.7 kb), and pTB3-3C (8.5 kb), estimate the percentage of cells that were re- all of which were introduced into B. subtilis sistant to Phleo. Plasmid analysis of 50 colo- 8G5 competent cells.
SEGREGATIONAL
PLASMID STABILITY
I
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FIG. 1. Segregational stabilities of pTB9 13 (A and C) and pTB3 derivatives (B and D) in B. subtilis strain 8G5. Stability assayswere performed in the absence of selective antibiotics at 37°C (A and B) or 47°C (C and D). Stabilities are expressed as the percentage of colonies resistant to the selective antibiotic (Phleo).
SegregationalStabilities of pTB913 Derivatives The segregational stabilities of the pTB9 13 and pTB3 series of plasmids were studied at 37 and 47°C. At 37°C pTB913 was stably maintained. A slight degree of plasmid loss was observed, however, upon insertion of the 1C and 3C DNA fragments (Fig. IA). In the absence of the MO, plasmid maintenance was considerably reduced with the largest plasmid, pTB3-3C (Fig. 1B). These results showed that at 37°C both the increase in plasmid size and the absence of the MO resulted in increased levels of plasmid instability. The plasmid stability assays at 47°C (Figs. 1C and 1D) gave a striking result: in contrast to the results obtained at 37°C all pTB9 13 derivatives, including those lacking the MO, were stably maintained at this temperature. As one possible cause of the high plasmid stability at 47°C we conceived that growth advantages of plasmid-free over plasmid-
containing cells, which might exist at 37°C would be absent or strongly reduced at 47°C. To study this possibility, temperature-shift experiments were performed. Two B. subtilis 8G5 (pTB3-3C) cultures were grown for about 60 generations, one at 37°C and the other at 47°C. Both cultures were then divided into two equal parts, one of which was shifted to 47°C and the other kept at 37°C. The fractions of plasmid-containing cells were determined at regular intervals after further incubation. The results of the stability assaysshowed that after the shift of the culture that had been preincubated at 37’C, plasmid-free cells accumulated with almost similar kinetics at 37 and 47°C (Fig. 2A). These results can be explained by growth advantage of plasmid-free over plasmid-containing cells at both 37 and 47°C (about 15% of the cells in the culture preincubated at 37°C were plasmid-free at the time of the shift). Support for this explanation was obtained in the following way. B. subtilis 8G-5 cells containing pTB3-3C were mixed with
74
OSRAM, VENEMA, AND BRON lOOf
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FIG. 2. The effect oftemperature shifts on the maintenance of pTB3-3C in B. subtilis. Cells were cultured for about 60 generations at 37 or 47°C under nonselective conditions, after which the cultures were separated in two equal parts. One part was maintained at 37°C and the other at 47°C. (A) Culture preincubated at 37°C; (B) culture preincubated at 47°C. Arrows indicate the temperature shift. (C) Cells containing pTB3-3C were mixed with plasmid-free cells. Overgrowth of plasmid-free cells was monitored at 37 and 47°C.
plasmid-free cells in a ratio of about 85:15. The mixed cultures were subsequently grown at either 37 or 47°C. The results of stability assaysof these mixed cell populations (Fig. 2C) confirmed that at both 37 and 47°C plasmid-free cells rapidly overgrew cells containing the plasmid. This result strongly supports the view that in the experiment shown in Fig. 2A the overgrowth of plasmid-free cells caused their accumulation at 37 and 47°C. Since the overgrowth by plasmid-free cells was even slightly faster at 47 than at 37”C, the observed stabilization of pTB9 13 derivatives at 47°C did not, apparently result from reduced differences in growth rates between plasmid-free and plasmid-containing cells at this temperature. The results obtained with the culture that was preincubated at 47°C are shown in Fig. 2B. In this culture, plasmid-free cells were neither present at the time of the shift nor
formed after continued incubation at 47°C. This confirmed the conclusion from Figs. 1C and 1D that pTB9 13 derivatives were stably maintained at 47°C. After the temperature of this culture was shifted to 37 “C, plasmid-free cells accumulated rapidly, however. Apparently, the preincubation of the cells at 47°C could not prevent the subsequent accumulation of plasmid-free cells at 37°C. Together, the results presented in this section indicate that the primary cause of pTB913 stabilization at 47°C was a lowering in the probability of segregating plasmid-free cells. Once such cells were formed, they appeared to outcompete plasmid-containing cells rapidly, however, at both 37 and 47°C. Plasmid ProJles and Copy Numbers
Plasmids using RCR generate ssDNA (te Riele et al., 1986; Gruss and Ehrlich, 1989)
SEGREGATIONAL * -+-+-
B
c
+
PLASMID STABILITY
P+-E+-F+
A -+-+
B
-
75
IN H .s~thr///.t C
D +-+-+-
E
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+ HMW
37%
47%
FIG. 3. Plasmid patterns in total DNA lysates from B. .sz~h/ili.s XC5 harboring pTB9 I3 or pTB3 derivatives. (+) and (-) indicate that the DNA preparations were incubated with endonuclease Sl or left untreated. Left panel, 37°C; right panel. 47°C. (A) pTB9 13: (B) pTB9 I3- 1C; (C) pTB9 I3-3C: (D) pTB3; (E) pTB3-1C; and (F): pTB3-3C. The positions of ssDNA (>) and HMW DNA are indicated.
and, under certain conditions, HMW DNA in B. subtilis (Gruss and Ehrlich, 1988, 1989; Viret and Alonso, 1987; Viret et al., 1991). To investigate whether such replication products were also formed in B. subtilis under the different temperature conditions used in the present studies, total DNA lysates extracted from the various plasmid-containing cells were analyzed using Southern hybridizations. The results (Fig. 3) showed that ssDNA and HMW DNA were indeed present in certain plasmid preparations. However, no large differences were observed between the plasmid profiles from cultures grown at 37 or 47°C. For instance, significant amounts of ssDNA were found in the absenceof the MO, in particular with the smaller plasmids (Fig. 3, lanes D), but the relative amounts of ssDNA were roughly similar at 37 and 47°C. Also, small amounts of plasmid HMW DNA were present with plasmids carrying the 1C fragment (Fig. 3, lanes B), but the relative amounts of this DNA were about the same at 37 and 47°C. It proved difficult to produce clear hybridization profiles of the plasmids carrying the 3C insert: aspecific degradation of the plasmid DNA was consistly observed. Despite this difficulty, it was clear that with these plasmids large amounts of plasmid DNA were present at the HMW position (Fig. 3, lanes C and F) and that, again, there
were no striking differences between the results obtained at 37 and 47°C. Basedon these results, we concluded that the temperature (37 or 47°C) had no drastic effect on the amounts of ssDNA and HMW DNA relative to the other plasmid forms. Therefore, it is unlikely that gross alterations in the production of ssDNA and HMW plasmid DNA caused the high stability of pTB913 derivatives at 47°C. We next studied whether the temperature affected the copy numbers of the circular plasmid monomers (the amounts of circular multimeric molecules were low). Although it was difficult to reproduce the copy number estimations quantitatively, we were able to determine the relative differences in copy numbers in a reproducible manner (three independent experiments were carried out). The averaged relative copy numbers per chromosome equivalent (Fig. 4) appeared to be reduced by either the insert (lC, about twofold; 3C, about fourfold) or by the absence of the MO (about twofold). In contrast, the relative copy numbers per chromosome equivalent were increased by a factor of about two when the temperature was raised from 37 to 47°C. In the method used here for the copy number determinations per chromosome equivalent, the eventual presence of linear HMW plasmid DNA, which migrates
76
OSKAM, VENEMA, AND BRON
;miii
pTE313 plTB13-1c pTE!P13-x plP3 t%sm!d
pTB3-1c
pm3-3c
FIG. 4. Relative plasmid copy numbers in B. subtilis 8G5 at 37 and 47°C. Copy numbers of the monomeric circular plasmid forms were determined per chromosome equivalent. The entries in the figure, presented as relative copy numbers, were the average ofthree independent experiments. As a standard, the copy number of pTB9 I3 at 37°C was set at 100%:this represents an actual copy number of approximately 30 per chromosome equivalent.
DNA inserts and the removal of the MO resulted in reductions of the plasmid copy numbers. Random partitioning of the available plasmid copies would then generate plasmidfree cells at a higher frequency. In agreement with this explanation was the observation in the present work that increased plasmid size or deletion of the MO resulted in reduced copy numbers. We can conceive of at least four possible causesfor the high segregational stability observed with pTB913 derivatives at 47°C: (i) effects of a heat-shock response, (ii) effectson ssDNA and HMW plasmid DNA formation; (iii) reduced differences in growth rates between plasmid-free and plasmid-containing cells, and (iv) increased copy numbers. These possibilities are discussed in the following sections. (i) Effectsof a Heat-Shock Response
together with the chromosal DNA fraction (Gruss and Ehrlich, 1988) might result in underestimates of the plasmid copy numbers. Since we estimated that the amounts of HMW plasmid DNA observed in the present studies were at most 10% of the chromosomal DNA (with plasmid pTB913-3C), we did not correct for possible errors due to HMW plasmid DNA.
Although no experimental evidence is available, one might speculate that in some unknown way the induction of heat-shock proteins was involved in the plasmid stabilization in B. subtilis at 47°C. In B. subtilis an impressive heat-shock response, involving as many as 66 proteins, has been observed (Miller et al., 1991). Moreover, it is known that some heat-shock-induced genes are involved in the replication of E. coli plasmids (Tilly and Yarmolinski, 1989; Kawasaki et DISCUSSION al., 1990). A role of heat-shock proteins in In the present work we studied the effects plasmid replication and plasmid stability in of the growth temperature of B. subtilis on B. subtilis remains to be established, howthe maintenance of pTB913, an RCR plasever. mid originating from a thermophilic Bacillus. The most striking result was that all (ii) Eflhcts on ssDNA and HMW Plasmid pTB9 13 derivatives tested were stably mainDNA Formation tained at 47°C although several derivatives Based on several studies it has been sugwere segregationally unstable at 37°C. Two parameters appeared to reduce the gested that the accumulation of ssDNA maintenance of pTB9 13 derivatives at 37°C: (Bron et al., 1985, 1988, 1991; Gruss et al., (i) the absence of a functional MO, and (ii) 1987; Viret and Alonso, 1987) or HMW increased plasmid size. These results are remi- DNA (Gruss and Ehrlich, 1988, 1989; Viret niscent of those described previously for et al., 1991) may reduce plasmid maintepUBll0 (Bron and Luxen, 1985; Bron et al., nance. The lower growth rates of plasmid1988, 1991). One possible explanation for containing compared to those of plasmidthese observations is that both the cloning of free cells, resulting in the overgrowth of the
SEGREGATlONAL
PLASMID
latter, have been considered as a likely cause of the reduced plasmid stability. In the present studies we showed that at 37°C reduced plasmid maintenance was associatedwith increased amounts of ssDNA (absence of the MO) or HMW DNA (presence of DNA inserts). This leaves the possibility open that at 37°C these replication products contributed to plasmid instability. At 47”C, the relative amounts of ssDNA and HMW DNA formed by pTB913 derivatives were, however, roughly similar to those at 37°C. This means that the plasmid stabilization observed at 47°C was not the consequence of a reduced accumulation of ssDNA or HMW DNA. Clearly, the considerable amounts of ssDNA and HMW DNA that accumulated at 47°C with some pTB9 13 derivatives did not reduce plasmid maintenance at this temperature. This could mean that the general idea that ssDNA/HMW DNA interfere with plasmid maintenance is incorrect or, alternatively, that specifically at the elevated temperature the harmful effects of these replication products are reduced. If the latter possibility is true, then one could speculate that the heatshock response described in the foregoing section plays a role in this process.
STABILITY
IN L1. .mh/i/i\
17
(iv) Increased Copy Numbers
We consider this the most attractive explanation for at least part of the observed plasmid stabilization effects at 47°C. The pTB913 copy numbers per chromosome equivalent were approximately twofold higher at 47 than at 37°C. This corroborates results by Shivakumar and Dubnau ( 1978), which indicated that at 47°C the amount of pUBll0 DNA was increased about twofold in B. subtilis. The increased copy numbers are expected to reduce the probability of generating plasmid-free cells. Copy number levels cannot, however, explain all observations in the present studies. For instance, plasmid pTB9 13- 1C showed a low, yet distinct degree of instability at 37”C, while plasmid pTB33C was fully stable at 47°C. Nevertheless, the copy number of pTB9 13-1C at 37°C was higher than that of pTB3-3C at 47°C. The nature of the putative additional factor increasing the stability of pTB9 13 plasmids at 47°C is not clear. As already discussed, we consider the heat-shock response a possible candidate for this function. The cause of the increased copy numbers at 47°C is a matter of speculation. Apparently, the copy control system enables the establishment of higher plasmid levels at 47°C. (iii) Reduced Diferences in Growth Rates Several explanations can be entertained for betweenPlasmid-Free and Plasmidthis. One is that at the higher temperature the Containing Cells interaction between the replication origin Growth advantage of plasmid-free over and the rate-limiting replication initiation plasmid-containing cells can be an important protein is more efficient, This might, for infactor in plasmid instability in bacteria (Bee stance, be due to altered sup&&city of the et al., 1987). This also appeared to be the case plasmid DNA at 47°C. Gennaro and Novick in the studies with pTB9 13 reported here, (I!%@), who analyzed the RCR plasmid Once formed, plasmid-free c&s appeared to pT 181, have indicated that the interaction beoutcompete plasmid-containing cells rapidly, tween the origin and the replication protein is at both 37 and 47°C. Reduced di&rew in imp&ant for rapid readjustments of the growth rates between plasm&free and p& copy number after, for instance, cell division. mid-containing cells cannot, therefore, ex- In out ,,iiem, a more efficient interaction plain the observed plasmid stabilization at between the pTB913 origin and the replica47°C. Our results rather indicated that the tion protein at 47°C might enable more stabilization of pTB9 13 derivatives at 47Y rounds of plasmid replication during the was due primarily to events that reduced the short life cycle of the cell, which lasts for only probability of segregating plasmid-free cells about 20 min at 47°C. The second possible from plasmid-containing parents. explanation is that more replication initia-
78
OSKAM,
VENEMA,
tion protein is produced at the higher temperature. This could be mediated through an altered efficiency of interaction of pTB9 13 antisense RNAs with the mRNA specifying the replication initiation protein. It has been shown that antisense RNA is important in the regulation of the copy number of plasmid pT 181 (Kumar and Novick, 1985). For pLS1 (Perez-Martinetal., 1988)andpUBllO(Maciag et al., 1988) which is very similar to pTB9 13, regulation of copy numbers via antisense RNA has been proposed. This makes it attractive to believe that antisense RNA also plays a role in the regulation of pTB9 13 copy numbers. In conclusion, these studies showed that in B. subtilis the stable maintenance of pTB9 13 derivatives can be realized by growing the cell cultures at elevated temperatures. This is a valuable property for the development of this plasmid as a cloning vector for bacilli. We have already extended our studies to thermophilic bacilli: in certain strains of Bacillus stearothevmophilus,pTB9 13 derivatives appeared to be fairly stable up to at least 57°C (unpublished results). So far, we have not tested whether RCR plasmids other than pTB913 are stably maintained at elevated temperatures, but, given the high degree of relatedness between various RCR plasmids (Gruss and Ehrlich, 1989) we consider this a likely possibility. ACKNOWLEDGMENTS This work was supported by the Program Commission Industrial Biotechnology of the Ministry of Economic Affairs (The Netherlands). We thank Jan Maarten van Dijl for valuable discussions, Arie Hamminga for preparation of the media, and Henk Mulder for preparing the figures.
REFERENCES BOE, L., GERDES, K., AND MOLIN, S. (1987). Effect of genes exerting growth inhibition and plasmid stability on plasmid maintenance. J. Bacteriol. 169, 46464650. BRON, S. (1990). Plasmids. In “Molecular Biological Methods for Bacillus” (C. R. Harwood and S. M. Cutting, Eds.), pp. 75-174. Wiley, Chichester, UK. BRON, S., BOSMA, P., VAN BELKUM, M., AND LUXEN, E.
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