FEMSMicrobiologyLoners9.'1(1992)19-22 © 1992Federationof EuropeanMicrobiologicalSocieties0378-1007/92/$05.00 Publishedby Elsevier
FEMSLE049t6
Electrotransformation of the stable L-form of Proteus mirabilis Ulrich Katenkamp a, Ingrid Groth a, Frank Laplace b and Horst Malke b 'J Hans-Kn~ll.bvstitutfiir Nalurstoff-Forschung. and h lnstitut fi'~rMolekularbiologieder Unit'ersitiitJena. ,lena. FRG
Received24 February1992 Revisionreceived25 March 1992 Accepted25 March 1992 Key words: Proteus mirabilis; Dform; Electrotransformation 1. SUMMARY To improve the t:ansformability of stable protoplast type L-forms of Proteus mirabilis for recombinant plasmid DNA, conditio~,~ for efficient electrotransformation were expt~,fed. Exposing cells from the exponential phase of growth at a density of 6-8 × 109/ml in electrotransformation buffer having a conductivity of 1.4 mS/cm to a field strength of 6.5 kV/cm for a mean pulse duration time of 1.2 ms reproducibly yielded transformation efficiencies in the order of 5 x 104 transformants per/~g of DNA. Compared to the polyethylene glycol method for transformation, electrotransformation appeared to be the method of choice for introduction of plasmid DNA into L-form cells. 2. INTRODUCTION Strain LVI derived from Proteus mirabilis is a stable protoplast type L-form that lacks any organized constituents of the peptidoglycan layer and no longer reverts to the parental bacterial form when serially passaged on solid or in liquid anCorrespondence to: H. Malke, lnstitut for Motekularbiologie
der Universit~itJena, Beutenbergstr.11, D-6900Jena, FRG.
tibiotic-free media of normal osmolarity [1,2]. Genetically engineered LVI derivatives have been constructed in our laborato~ to yield recombinant DNA products in biologically active forms at high yields [3,4]. These results, together with evidence for correct heterologous signal sequence processing [5], suggest that LVI may be generally useful as an alternative organism for the production of recombinant proteins in secreted form at the laborato~ scale. A major obstacle to laboratory utility has been the low and erratic transformation frequency when plasmid DNA was introducet~ into the cells by conventional methods using polyethylene glycol (PEG) in the presence of sucrose at high concentrations [3]. To improve transformability, we have now systematically used electrotransformation for the introduction of plasmid DNA into LVI and define here optimum conditions for reproducibly achieving high transformation efficiencies. 3. MATERIALS AND METHODS 3.1. Bacterial strains, plasmids and culture contritions
The shaken 100-ml extract
stable L-form strain LVI was grown in flasks (250 rpm; 30 ml of culturt: in a flask) at 37"C in freshly prepared beef supplemented with Bacto peptone, yeast
extract (Difco), sucrose and NaCI, as described [3]. The parent strain, Proteus mirabilis VI, was grown aerobically in LB medium [6]. For growth on agar, the liquid media were solidified with 1.5% agar (Difco) and, if required, erythromycin was added at 10 /zg/ml. The time course of growth in liquid media was followed by determining the optical density at 560 nm. The recombinant plasmid pMLS10 (7.3 kb) used in the transformation experiments is a shuttle vector carrying an erythromycin-resistance determinant and the streptokinase gene, skc [5].
3.2. Electrotransformation L-form cells from different phases of the growth cycle were harvested by centrifugation (10 rain at 700 X g) and washed twice with electrotransformation buffer containing 0.3 M sucrose, 7 mM sodium phosphate, and 1 mM MgCi 2 (pH 7.4; conductivity, 1.4 mS/cm). For normal cells, this buffer solution was diluted with water to a conductivity of 0.5 mS/cm. After washing, the bacteria were resuspended in their respective buffers at 6-8 x 109 cells/mi. Electroporation was carried out at variable parameter settings with the Bio-Rad Gene Pulser equipped with Pulse Controller and Capacitance Extender. 100 #1 of the cell suspensions were mixed with 10 #1 of plasmid DNA (approx. 1 ,ttg/ml), transferred to chilled cuvettes (2-mm electrode gap) and exposed to single electric pulses (1.2 and 5 ms for L-forms and normal cells, respectively) at electric field strengths ranging from 2 to 11 kV/cm. After treatment and storage on ice for 10 rain, the cells were mixed with 900/~1 of growth medium and incubated for 2 h at 37°C before being plated on selective medium to develop erythromycin-resistant colonies. L-form colonies appearing after 3-4 days of incubation were overlayed with streptokinase assay medium [7] to test for the presence of the skc gene. 4. RESULTS AND DISCUSSION
4.1. Growth characteristics of normal and L-form cells To assign cells to be electrotransformed to specific growth phases, the time course of L-form
growth was compared to that of the normal form by OD measurements. Whereas the growth of the normal form followed the typical exponential course before becoming stationary, L-form cultures grew more irregularly, with strict exponential growth being restricted to the first 4-6 h of the growth cycle. After a pronounced quasi-stationary phase of about 4 h, growth resumed but was characterized by periodic decreases followed by increases of the OD curve. This overall pattern of growth in liquid medium may be explained by phases of cell lysis followed by renewed growth and may further be a reflection of unequal cell division processes characteristic of the L-form state. Phase contrast microscopy of cells from the principal phases of growth revealed differences in size as well in shape. In particular, L-form cells from the stationary growth phase appeared to be stressed in that they developed lobes, in contrast to the spherical appearance of log-phase cells.
4.2. Field sensitivity Electric field sensitivity was studied next for normal and L-form cells from mid-log and stationary cultures. Cells of the normal bacterial form when pulsed for 5 ms survived increasing field strengths much better when coming from the stationary phase rather than from log-phase cultures. For the former, a survival rate of 50% was obtained at about twice the field strength required for the latter (E = 9.3 and 4.8 kV/cm, respectively). In contrast, L-form cells pulsed for 1.2 ms were about equally sensitive to the electric field regardless of the culture age they represented. Irreversible permeabilization of the cell envelopes occurred in 50% of either cells at a field strength of 5-6 kV/cm. 4.3. Electrotransformation Initial experiments designed to introduce pLMS10 into L-form cells by electrotransformation were carried out with stationary phase cells at various parameter settings. Similar to PEGtreated organisms, these cells yielded highly variable transformation frequencies ranging from < 10 to 103 transformants per fig of DNA. In contrast, transformation of log-phase cells as a function of the electric field strength yielded re-
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Fig. !. Transformability and field sensitivity of exponentially growing L-form cells as a function of field strength. For L-form cultures, OD measurement carried out 2 h after the pulse was found to be a more reliable measure of field sensitivity than the counting of colony-forming units. Cell survival and transformability of c :l,s transformed by the standard PEG method [3] a.-; aho indicated.
producible results, as shown in Fig. 1 for a representative experiment. There was a well-defined optimum range of the field strength for transformation, with the peak of transformability (5 x I04 transformants/~g DNA) occurring at E = 6.5 kV/cm. The maximum number of transformants was obtained at a survival frequency of about 30% (Fig. 1), corresponding to a transformation rate (i.e., transformants/survivors) of about 2 × 10 -4 . For comparison, log-phase cells transformed by the PEG method [3] gave transformation yields about two orders of magnitude lower than those obtained using optimum electrotransformation conditions (Fig. 1). Interestingly, about the same relationship between optimal electrotransformability and cell survival holds for a wide range of organisms including Escherichia coli [8], Streptococcus [9] and Mycobacterium [10]. It is generally accepted that the field strength to be applied for membrane poration depends only on the cell radius, assuming a critical interfacial potential difference of about 1 V [11]. Since among prokaryotes the cell radius varies relatively little, this may explain the narrow range of field strength (5-10 kV/cm) suitable to electrotransform bacteria. In contrast to this, the pulse duration required for optimum transformation seems to be strongly influenced by
the nature of the cell envelope. The available data show that time constants may range from 1 ms in the case of L-forms featuring a protoplasttype envelope to longer than 10 ms for myeobacteria characterized by sturdy cell walls rich in lipids [10], In the case of L-forms, unequal cell division gives rise to differently sized cells, suggesting that using subpopulations of L-form cells with a more uniform cell diameter may increase and further stabilize their electrotransformation rate. For practical purposes, it would appear that under the present optimum conditions defined by bo,h biological and electrical parameters, electrotransformation is the method of choice for the introduction of plasmid DNA into L-form cells.
ACKNOWLEDGEM ENTS We thank Bio-Rad Laboratories for enabling us to use their Gene Pulser in these experiments. This work was supported by Fonds der Chemischen lndustrie (400126) and Deutsche Forschungsgemeinschaft (Ma 1330/1-1).
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131 Klessen,C.. Schmidt. K.-H., Gumpert+J., Grosse, H.-H. and Malke, H. (1989). Appl. Environ. Microbiol. 55, 1009-10f5. [4] Laplace, F., Miiller, J., Gumperl, J. and Malke, H. (1989) FEMS Microbiol. Lett. 65, 89-94. [5] Laplace, F., Egerer, R., Gumpert, L, KraR, R., Kostka, S. and Malke, H. {1989) FEMS Microbiol. Left. 59, 59-64. [61 Lennox, E.S, (1955) Virology I, 19{I-206, [7] Malke, H. and Ferretti, J.J. (1984) Proe. Natl. Aead. Sci. USA 81, 3557-3561. [8] Wirth, g+, Friescnegger, A, and Fiedler, S. (1989) Moi. Gen+ Genet. 216, 175-177. [9] Simon, D. and Ferrelti, J.J. (1991) FEMS Microbiol. Lett. 82, 219-224. [10] Katenkamp, U., Atrat, P. and Huller, E+ 110911 Bioelectrochem. Bioenergetics 25, 285-294. [ll] Neumann, E., Sowers, A,E. and Jordan, C.A. (1989) Electroporation and Electrofusion in Cell Biology. Plenum Press, New York and London.