FEMS Microbiology Letters 37 (1986) 309-312 309 Published by Elsevier

FEM 02611

The effect of procaine on the partitioning of plasmid pSC101

(Procaine; plasmid partitioning; Escherichia coli; phosphate-limited chemostat)

Mark Lancaster , Susan Prior and Nicholas H. M a n n *

Department of Biological Sciences, University of Warwick, Coventry CV4 7A L, U.K.

Received and accepted 1 September 1986

1. SUMMARY

The effect of procaine at a sub-lethal con- centration (20 mM) on the segregational stability of pSC101 and other plasmids in Escherichia coli was examined in phosphate-limited chemostat cul- ture. Procaine caused segregational instability of pSC101 and a derivative of pBR322 carrying the par function of pSC101. The segregational stabil- ity of 2 other unrelated plasmids, RP4 and pDS1109, was unaffected by procaine. It is con- cluded that the synthesis of a chromosomally en- coded membrane protein, under the control of the enoZ gene product, is required for pSC101 par function or that the functioning of this protein is impaired by the presence of procaine.

2. INTRODUCTION

The active partitioning process involved in the stable maintenance of plasmid pSC101 has been shown to involve a cis-acting locus designated par which has been located in a 270 bp segment adjacent to the origin of replication [1]. The se- quence of par [2,3] reveals no open reading frames, but does contain three segments, designated a, b and a', which are potentially capable of forming stem-loop structures. Deletion studies have con- firmed the importance of these segments in the

partitioning process of pSC101 [4]. Gustafsson et al. [5] have shown that par + plasmids can bind to a membrane fraction of the same buoyant density as the outer membrane, whilst p a r - plasmids do not. Since pSC101 does not appear to encode any trans-acting factor involved in partitioning [2], the protein(s) involved in this membrane site must be host-encoded.

The administration of sub-lethal concentrations of procaine to E. coli causes pleiotropic alterations in the synthesis of a wide variety of outer mem- brane and periplasmic proteins [6-10]. We were interested in establishing whether sub-lethal con- centrations of procaine would interfere with the stable maintenance of pSC101 by inhibiting the synthesis of a chromosomally encoded membrane protein required for par function. Since pSC101 is present at a sufficient copy number (approx. 8) to ensure that even if active partitioning were abolished, the appearance of plasmid-free cells might be a rare event, we decided to investigate the effect of procaine on the integrity of the pSC101 partitioning process and that of other stably maintained plasmids in phosphate,limited chemostat culture. Growth of plasmid-carrying strains and their plasmid-free isogens in the chem- ostat under phosphate-limitation has been shown to offer a selective advantage to the plasmid-free isogen [11-13] and would thus facilitate the detec- tion of plasmid-free segregants.

0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies

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3. MATERIALS AND METHODS

3.1. Bacterial strains and plasmids The strain of E. coli used throughout this work,

ML3, (thr, |hi, strR), was derived from W5445 (pro, leu, thr, thi, str R) by P1 transduction. The plasmids used were pDSll09 (ApR), pSC101 (TcR), pWX9 (Ap g) and RP4 (ApRKmRTcR).

3.2. Chemicals Ampicillin (sodium salt), tetracycline hydro-

chloride, streptomycin sulphate and procaine hy- drochloride were obtained from Sigma. Where required, antibiotics were used at the following concentrations: ampicillin, 50/~g/ml; tetracycline, 15/~g/ml; streptomycin, 100/tg/ml. Procaine was used at 20 mM.

3.3. Media The minimal salts medium for phosphate-

limited chemostat culture was that of Jones et al. [11], except that it also contained ferric citrate 1.8 mg. ml 1 and was supplemented with threonine (50 /xg.m1-1) and thiamine (10 /~g. ml-1). The nutrient medium was L-broth which when neces- sary was solidified by the addition of 1.5% (w/v) Bacto agar.

3.4. Chemostat culture Cultures to be used as inocula for the chem-

ostat were grown overnight at 37°C in L-broth, containing the appropriate selective antibiotic, Chemostat cultures were grown in an LH 500 Series 3 fermenter (LH Engineering). Culture pH was maintained at 7.0 using 1M NaOH and the growth temperature was 37°C. Oxygen tension was monitored and was maintained at above 70% saturation at all times. The working volume of the fermenter was 500 ml, and in all cases the dilution rate (D) was 0.2 h -1.

3.5. Screening for plasmid-free segregants Samples were removed from the chemostat at

intervals, appropriately diluted, and plated on L- agar containing streptomycin. 100 colonies from each sample were patch tested on plates contain- ing the appropriate antibiotic. Antibiotic-sensitive clones were tested for the presence of the plasmid

by a small scale plasmid extraction procedure [14] and subsequent electrophoresis in 1% (w/v) agarose gels.

3.6. Transformation Transformations of E. coli was done by the

method of Holland [15].

4. RESULTS AND DISCUSSION

E. coil ML3 carrying plasmid pSC101 was maintained in the chemostat under phosphate limitation for 135 generations, during which time no plasmid-free segregants were detected (Fig. 1). However, when the experiment was repeated using medium containing 20 mM procaine, 42% of the population were tetracycline-sensitive after 140 generations of growth (Fig. 1). Tetracycline-sensi- tive clones were screened for the presence of plasmid DNA; 62.5% (10/16) contained no detec- table plasmid, while the remaining clones con- tained plasmid DNA which migrated slightly fas- ter than authentic pSC101 on agarose gel electro-

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Fig. 1. Segregational stability of pSC101 in E. coil ML3 grown in phosphate-limited chemostat culture (D = 0.2 h - l ) . The proportion of plasmid-containing cells was determined for cultures grown (O) in the absence, and (O) in the presence of 20 mM procaine.

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Fig. 2. Competition between pSC101-containing and plasmid- free E. coli ML3 in phosphate-limited chemostat culture in the presence of 20 mM procaine (D = 0.2 h - l ) . The chemostat was inoculated with either (©) pSC101-containing cells; or ( , ) pSC101-containing cells plus a 1% inoculum of plasmid- free cells.

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Fig. 3. Segregational stab!!ity of pWX9 in E. coli ML3 grown in phosphate-limited chemostat culture ( D = 0.2 h 1). The chemostat was inoculated with (z~) cells containing monomeric pWX9 in the absence of procaine; (~,) cells containing mono- meric pWX9 in the presence of 20 mM procaine; and (v) cells containing dimeric pWX9 in the presence of 20 mM procaine.

phoresis and presumably represented deletion de- rivatives of pSC101. When a mixture of pSC101- containing and plasmid-free cells (ratio 99 : 1) was inoculated into the chemostat and maintained in the presence of 20 mM procaine, the plasmid-free isogen took over the culture, and after 135 genera- tions represented nearly 80% of the population (Fig. 2). To establish whether the cells which had lost pSC101 were mutants defective in plasmid maintenance, a plasmid-free colony derived from the chemostat was transformed with pSC101 and then subjected to another round of chemostat culture in the presence of procaine. The pattern of plasmid loss (data not shown) was similar to that of the parental culture and consequently it can be assumed that plasmid-free cells arising in the pres- ence of procaine do not carry mutations affecting plasmid maintenance.

Plasmid pWX9 is a derivative of pBR322 which contains the E c o R I - A v a I par fragment of pSC101 replacing the normal E c o R I - A v a I segment [16], and is reported to be segregationally stable in phosphate-limited chemostat culture [16]; this was

confirmed in this study (Fig. 3) with no ampicil- lin-sensitive cells being detected after about 140 generations of phosphate-limited growth. In con- trast, when E. coli ML3[pWX9] was maintained in the chemostat in the presence of 20 mM procaine, plasmid-free cells were detected after about 20 generations of growth. When a dimeric form of pWX9, which should exist at half the copy num- ber of the monomer, was introduced into ML3, instability was enhanced, with about 40% of the population being ampicillin-sensitive after 130 generations.

To determine whether the effect of procaine on plasmid partitioning was specific for par function of pSC101, its effect on the stability of 2 other plasmids was examined, pDSll09 is a Tn3 deriva- tive of the high copy number plasmid ColE1 in which colicin E1 production is inactivated by the Tn3 insertion [17]. RP4 is a broad host-range, low copy number conjugative plasmid, pDSl l09 proved to be completely stable during prolonged phosphate-limited chemostat culture in the pres- ence of 20 mM procaine with no plasmid-free

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segregants being detected after 150 generations (Fig. 4). Similar stability with respect to procaine was observed for RP4 (data not shown). When a mixture of pDSll09-containing cells and plasmid- free cells (99:1) was used as an inoculum the plasmid free cells rapidly took over the chemostat in the presence of procaine (Fig. 4). This confirms that had any plasmid-free cells arisen when only plasmid-carrying cells were used as an inoculum, they would have been detected.

It seems likely therefore that procaine is in- hibiting the synthesis of a chromosomally-encoded membrane protein which is required to interact with the par function of pSC101 to permit active partitioning. The effects of procaine mimic one class of mutation in enoZ [18], typified by the envZ473 allele. Other classes of mutation in envZ, represented by the enoZ3 [18], envZ6 [18] and envZ amber [19] mutations abolish the pleiotropic effects of procaine, suggesting that its action may not be mediated by general membrane structural alterations, but rather by a specific interaction, possibly with the envZ gene product itself [18,19].

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Fig. 4. Competition between pDSll09-containing an/d plasmid-free E. coli ML3 in phosphate-limited chemostat cul- ture in the presence of 20 mM procaine (D = 0.2 h - l ) . The chemostat was inoculated with either (El) pDSll09-containilag cells; or (11) pDSll09-containing cells plus a 1% inoculum lof plasmid-free cells.

Consequently, it would appear that the synthesis of a membrane protein or proteins involved in pSC101 partitioning is under the control of the envZ gene product, or that the functioning of this protein is impaired by the presence of procaine.

ACKNOWLEDGEMENTS

This research was supported by grant number GR/C66155 from the Science and Engineering Research Council (U.K.).

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