Optimization of Protoplast Formation and Regeneration in ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1987, p. 1694-1695 0099-2240/87/071694-02$02.00/0 Copyright C) 1987, American Society for Microbiology

Vol. 53, No. 7

Optimization of Protoplast Formation and Regeneration in Leuconostoc mesenteroides DAVID R. OTTS AND D. F. DAY* Department of Microbiology and Audubon Sugar Institute, Louisiana State University,

Baton Rouge, Louisiana 70803-7305 Received 17 February 1987/Accepted 2 April 1987

Conditions are reported for efficient protoplast formation and regeneration in four strains of Leuconostoc mesenteroides. Protoplasts were produced from each strain at frequencies greater than 99%, although the rate of their production was variable from strain to strain. Bovine serum albumin and Mg2+ were required for maximal regeneration, while the presence of Ca2+ was inhibitory. Regeneration frequencies of 16% could be obtained with strain ATCC 10830. This frequency was four- to eightfold higher than the frequencies of the other strains examined.

The ability of Leuconostoc mesenteroides to produce the polysaccharide dextran from sucrose has plagued the sugar industry for years (2). The enzyme responsible for this conversion, dextransucrase, has been studied extensively over the past 40 years (1, 3, 9-11). Although much is known about this enzyme, surprisingly little is known about the regulation of its production (5, 7, 12). In general, the study of molecular regulation in L. mesenteroides has not been feasible because of the lack of suitable genetic exchange systems. For this reason, we have examined the conditions required for the production and regeneration of protoplasts of L. mesenteroides with the aim of establishing a protoplast transformation system. (This work was presented in part at the 86th Annual Meeting of the American Society for Microbiology, Washington, D.C., 23 to 28 March 1986). L. mesenteroides ATCC 10830, ATCC 8293, ATCC 14935, and DR203, a strain isolated from Louisiana sugarcane, were used in this study. Strains were maintained on All Purpose Tween agar and broth (Difco Laboratories, Detroit, Mich.). Leuconostoc medium plus fructose (LMF) consisted of 1.0% tryptone, 0.7% yeast extract, 0.5% K2HPO4, and 1.0% fructose (pH 7.0). Regeneration medium consisted of LMF supplemented with 0.5% bovine serum albumin, 25 mM MgCl2, and 19% (wt/vol) maltose. Bovine serum albumin was prepared by heat inactivation of a 10% (wt/vol) solution at 55°C for 30 min followed by filter sterilization through a membrane filter (0.22-p,m pore size; Millipore Corp., Bedford, Mass.). Fructose, bovine serurm albumin, and MgCl2 were added to regeneration medium after the medium was autoclaved. Protoplast buffer consisted of 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.0), 1 mM MgCl2, and 19% (wt/vol) maltose as the osmotic stabilizer. To prepare protoplasts, cells were cultivated in All Purpose Tween broth overnight at 30°C. Then 50 ml of fresh All Purpose Tween broth was inoculated with 5 ml of this culture, and incubation was continued at 30°C until the A660 reached approximately 0.5. At this point, cells were harvested by centrifugation, washed once with protoplast buffer, and suspended in 5 ml of the buffer. The cell concentration at this point was approximately 4.0 x 108 CFU/ml. Cells were allowed to equilibrate at 37°C for 5 min, *

and then 10 mg of lysozyme (14,400 U/mg) was added. The suspension was incubated at 37°C with periodic gentle mixing. Protoplast formation was monitored by phase-contrast imicroscopy and by the decrease in the number of colonies obtained on LMF agar after dilution in distilled water. After treatment with lysozyme, protoplasts were harvested by centrifugation at 2,500 x g for 15 min. They were carefully resuspended in 5 ml of protoplast buffer and then recentrifuged. This washing procedure was repeated twice, with the final pellet resuspended in 5 ml of protoplast buffer. Both regenerated protoplasts and nonprotoplast cells were enumerated by dilution of this suspension in protoplast buffer and plating on regeneration medium. Nonprotoplast cells were enumerated by dilution in distilled water and plating on LMF agar. All plates were incubated at 30°C and counted after 5 days. The regeneration frequency was the ratio of regenerated protoplasts to protoplasts produced, i.e., (initial cell number - nonprotoplast cells) x 100. L. mesenteroides strains varied greatly in their degree of sensitivity to lysozyme (Fig. 1). Protoplast efficiencies greater than 99.9% could be obtained from all strains. Cells of strain ATCC 10830 clumped within 5 min after the addition of lysozyme. This clumping could not be negated by increasing the magnesium concentration in the protoplast buffer. None of the other L. mesenteroides strains examined demonstrated the ability to clump in protoplast buffer. The effect of lysozyme digestion time on the regeneration of L. mesenteroides is shown in Table 1. Lengthy treatment with lysozyme decreased the ability of the protoplasts to regenerate. For this reason, digestion times were kept to a minimum with each strain. Times were chosen for each strain so that protoplast efficiencies of less than 99.9% were obtained. Regeneration of L. rfesenteroides strains with spread plates was complete in 5 days. The use of soft-agar overlays did not significantly increase the regeneration frequencies of the strains examined. However, regeneration was faster with the overlays, as regeneration was complete after 3 days. The effects of substitution of different regeneration medium components on the regeneration of L. mesenteroides strains are shown in Table 2. The addition of calcium at a cOncentration of 25 mM depressed regeneration of all strains examined. Removal of magnesium from the regeneration medium also inhibited regeneration. All strains showed a strong requirement for bovine serum albumin for optimal

Corresponding author. 1694

VOL. 53, 1987

NOTES

Strain

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TABLE 2. Effect of substitution of regeneration medium components Regeneration frequency in medium':

ATCC 10830 ATCC 14935 ATCC 8293 DR203

1

2

3

4

5

6

16.0 4.0 2.0 3.5

0.07 0.25 0.01 0.22

14.0 4.2 1.0 2.5

1.7 2.0 0.07 0.44

2.4 1.0 0.23 0.25

16.0 3.0 2.0 3.5

aMedium 1 was the regeneration medium described in the text. Media 2 through 6 are medium 1 lacking (-) or containing (+) the following components: 2, bovine serum albumin (-); 3, 2.5% gelatin (+); 4, 25 mM MgCl2 (-); 5, 25 mM CaCl2 (+), MgCI. (-); 6, 0.5 M lactose (+), 0.5 M maltose (-).

This strain clearly has potential for establishing a protoplast transformation system. Regeneration frequencies obtained in this study are comparable to those obtained with other lactic acid bacteria (4, 6). With the demonstration of plasmids in Leuconostoc spp. (8), rigorous attempts to develop a transformation system in this organism can now be made. INCUBATION TIME (minutos)

FIG. 1. Time course of the production of osmotically sensitive cells from L. mesenteroides strains. The curves represent data obtained for strains ATCC 10830 (0), ATCC 14935 (N), ATCC 8293 (0), and DR203 (A). Cells were incubated with 2 mg of lysozyme per ml as described in the text. At indicated times, cells were diluted in distilled water and plated on LMF agar.

regeneration, while the addition of gelatin at a concentration of 2.5% did not significantly affect regeneration frequencies. Lactose (0.5 M), but not raffinose (0.3 M) or sucrose (0.5 M), could effectively replace maltose as the osmotic stabilizer. The results of this study indicate that optimal conditions for the production and regeneration of L. mesenteroides protoplasts must be determined for the particular strain of interest due to strain-to-strain variability. Care must be taken not to overdigest the cells with lysozyme, as regeneration frequencies rapidly decline with prolonged digestion. Strain ATCC 10830 showed four- to eightfold-higher regeneration frequencies than did the other L. mesenteroides strains examined. The reason for this difference is unknown.

TABLE 1. Effect of lysozyme digestion time

ATCC 14935

regeneration

Protoplast formation

Regeneration frequency

5 20 60

99.9840 99.9972 99.9999

90

>99.9999

5 15 30 60

93.8200 98.6500 99.8240 99.9990

42.51 0.52 1.16 x 10-4 >1.00 X lo-4 8.62 5.25 2.04 0.19

Time (mm)

ATCC 10830

on

%

LITERATURE CITED 1. Hehre, E. J. 1946. Studies on the enzymatic synthesis of dextran from sucrose. J. Biol. Chem. 163:221-233. 2. Imrie, F. K. E., and R. H. Tilbury. 1972. Polysaccharides in sugar cane and its products. Sugar Technol. Rev. 1:291-361. 3. Kobayashi, M., and K. Matsuda. 1986. Electrophoretic analysis of the multiple forms of dextransucrase from Leuconostoc mesenteroides. J. Biochem. 100:615-621. 4. Kondo, J. K., and L. L. McKay. 1984. Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning. Appl. Environ. Microbiol. 48:252-259. 5. Lawford, G. R., A. Kligerman, and T. Williams. 1979. Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides. Biotechnol. Bioeng. 21: 1121-1131. 6. Lee-Wickner, L.-J., and B. M. Chassy. 1984. Production and regeneration of Lactobacillus casei protoplasts. Appl. Environ. Microbiol. 48:994-1000. 7. Neely, W. B., and J. Nott. 1962. Dextransucrase, an induced enzyme from Leuconostoc mesenteroides. Biochemistry 1: 1136-1140. 8. Orberg, P. K., and W. E. Sandine. 1984. Common occurrence of plasmid DNA and vancomycin resistance in Leuconostoc spp. Appl. Environ. Microbiol. 48:1129-1133. 9. Robyt, J. F. 1980. Mechanism of polymerization of dextransucrase, p. 43-54. In J. J. Marshall (ed.), Mechanisms of saccharide polymerization and depolymerization. Academic Press, Inc., New York. 10. Robyt, J. F., and S. H. Eklund. 1983. Relative, quantitative effects of acceptors in the reaction of Leuconostoc mesenteroides B-512F dextransucrase. Carbohydr. Res. 121:279286. 11. Robyt, J. F., and T. F. Walseth. 1979. Production, purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F. Carbohydr. Res. 68:95-111. 12. Tsuchiya, H. M., H. J. Koepsell, J. Corman, G. Bryant, M. 0. Bogard, V. H. Feger, and R. W. Jackson. 1952. The effect of certain cultural factors on production of dextransucrase by Leuconostoc mesenteroides. J. Bacteriol. 64:521-526.