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Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli
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Metabolic Engineering 7 (2005) 45–52 www.elsevier.com/locate/ymben
Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli MingHua Daia, Sara Ziesmanb, Thomas Ratcliffeb, Ryan T. Gillb, Shelley D. Copleya, a
Department of Molecular, Cellular, and Developmental Biology and Cooperative Institute for Research in Environmental Sciences, University of Colorado at Boulder, Boulder, CO 80309, USA b Department of Chemical and Biological Engineering, University of Colorado at Boulder, Boulder, CO 80309, USA Received 12 May 2004; accepted 13 September 2004 Available online 10 November 2004
Abstract Protoplast fusion has been used to combine genes from different organisms to create strains with desired properties. A recently developed variant on this approach, genome shuffling, involves generation of a genetically heterogeneous population of a single organism, followed by recursive protoplast fusion to allow recombination of mutations within the fused protoplasts. These are powerful techniques for engineering of microbial strains for desirable industrial properties. However, there is a prevailing opinion that it will be difficult to use these methods for engineering of Gram-negative bacteria because the outer membrane makes protoplast fusion more difficult. Here we describe the successful use of protoplast fusion in Escherichia coli. Using two auxotrophic strains of E. coli, we obtained prototrophic strains by recombination in fused protoplasts at frequencies of 0.05–0.7% based on the number of protoplasts subjected to fusion. This frequency is three–four orders of magnitude better than those previously reported for recombination in fused protoplasts of Gram-negative bacteria such as E. coli and Providencia alcalifaciens. r 2004 Elsevier Inc. All rights reserved.
1. Introduction Classical methods for improvement of microbial strains have relied upon either mutagenesis followed by selection for improved properties, or manipulation of specific genes known to play an important role in the desired phenotype. The first strategy is time-consuming, requiring many generations of mutation and selection to allow accumulation of multiple beneficial mutations in a single strain. The efficacy of the second strategy is limited by the ability to predict which mutations will improve a particular phenotype. Thus, it is not possible to take advantage of mutations in genes that are not obviously related to the phenotype of interest but may nevertheless improve microbial fitness or performance under a particular set of conditions. Genome shuffling is a recently introduced method that is much more efficient Corresponding author.
E-mail address: [email protected] (S.D. Copley). 1096-7176/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymben.2004.09.002
for evolution of strains of microbes with desirable phenotypes. It involves generation of a heterogeneous population of mutants by treatment with a chemical mutagen, followed by recursive fusion of protoplasts to allow recombination. This procedure allows rapid evolution of strains with multiple beneficial mutations. For example, recent reports have described the use of genome shuffling to improve production of tylosin by Streptomyces fradiae (Zhang et al., 2002), acid tolerance in Lactobacillus (Patnaik et al., 2002), and degradation of pentachlorophenol in Sphingobium chlorophenolicum (Dai and Copley, 2004). Other reports have described successful use of protoplast fusion (without the initial mutagenesis step) to combine metabolic capabilities of two different organisms. For example, protoplasts of Acinetobacter sp. A3 and Pseudomonas putida DP99 have been fused to generate strains with enhanced abilities to degrade hydrocarbons (Hanson and Desai, 1996), and protoplasts of Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15 have been fused to
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generate strains with an enhanced ability to produce sorbitol under fermentation conditions (Wei et al., 2001). The efficiency of genome shuffling is a function of the efficiencies of generation and fusion of protoplasts, of recovery of protoplasts after fusion, of recombination between heterogeneous chromosomes in the diploid or multiploid cells obtained after fusion, and of the eventual segregation of true prototrophs containing only one genome. Protoplasts are typically generated by treatment of cells with lysozyme to digest the peptidoglycan surrounding the inner membrane (Weiss, 1976; Hopwood, 1981). This process is quite efficient in Grampositive bacteria, but less so in Gram-negative bacteria because the outer membrane impedes access of lysozyme to the peptidoglycan layer. Indeed, it is a commonly held notion that protoplast fusion in Gram-negative organisms is too challenging for use as a strain engineering tool. This skepticism appears to be based on a small number of early studies reporting limited success in fusion of protoplasts from Gram-negative organisms. For example, Tsenin et al. (1978) reported generation of prototrophic colonies from fusion of protoplasts of two auxotrophs of Escherichia coli K12 at a frequency of only 1 10 5, and only 10% of these colonies appeared to be true prototrophs. Coetzee et al. (1979) reported generation of prototrophic colonies at frequencies ranging from 1.7 10 7 to 3.8 10 6 per protoplast originally present using auxotrophs of Providencia alcalifaciens strain P29. We have recently reported the use of genome shuffling in the Gram-negative bacterium S. chlorophenolicum to generate strains with an enhanced ability to degrade the toxic pesticide pentachlorophenol (Dai and Copley, 2004) and have begun to explore the use of genome shuffling in E. coli. Here we provide data demonstrating that the efficiency of recombination in fused E. coli protoplasts is lower than that achieved using Grampositive organisms, but is nevertheless high enough for practical applications. We have used electron microscopy to visualize the formation and fusion of protoplasts during the procedure, and have quantified the generation of prototrophic strains by recombination of parental genomes in fused protoplasts generated from two auxotrophic parental strains. These data provide encouragement for efforts to utilize genome shuffling in Gram-negative bacteria.
2. Materials and methods 2.1. Bacterial strains and growth conditions E. coli W4183: argG78, rpsL257, (strR), F , and E. coli FU20-1: leuB6(Am), phoA1, rpsL114(strR), malT1(lR), F were obtained from the E. coli Genetic
Stock Center (http://cgsc.biology.yale.edu). Cells were grown in either LB or M9 medium (Sambrook and Russell, 2001) supplemented with either arginine or leucine (100 mg/ml). All media contained streptomycin (10 mg/ml).
2.2. Protoplast formation Protoplasts were formed by modification of a previously reported method (Weiss, 1976). Each auxotroph was grown in 30 ml of LB medium containing streptomycin (10 mg/ml) until an OD550 of about 1.0 was reached. The cells were harvested by centrifugation at 3000g and 4 1C for 10 min, washed three times with 0.01 M Tris-HCl (pH 8.0), and resuspended in 30 ml 0.01 M Tris-HCl (pH 8.0) containing 0.5 M sucrose. Potassium ethylenediaminetetraacetate (EDTA, 0.1 M, pH 8.0) was added slowly over a period of 20 min to a final concentration of 0.01 M. The cells were shaken at 100 rpm at 37 1C for an additional 20 min to begin removal of the outer membrane. The cells were harvested by centrifugation at 3000g and 4 1C for 10 min and washed twice with SMM buffer (0.5 M sucrose, 20 mM sodium maleate, 20 mM MgCl2, pH 6.5). The cells were resuspended in 30 ml SMM buffer containing lysozyme (1 mg/ml). The cells were shaken at 37 1C at 100 rpm for 1 h to allow digestion of the peptidoglycan layer. For one experiment, cells were suspended in SMM buffer containing lysozyme (1 mg/ ml), and the cells were shaken at 100 rpm at 37 1C for 1 h. Subsequently, EDTA was added slowly to a final concentration of 0.01 M and the cells were shaken at 37 1C for 20 min. The efficiency of protoplast formation was judged by subjecting an aliquot of the cells to dilution in distilled water to lyse protoplasts, followed by plating of serial dilutions on LB agar to quantify colonies resulting from cells that had not formed protoplasts.
2.3. Protoplast regeneration Several conditions for regeneration of protoplasts were explored. In some cases, protoplasts were resuspended in LB medium containing 0.5 M sucrose and spread onto soft agar plates containing LB medium and 0.5 M sucrose with autoclaved plastic spreaders. The plates were subsequently incubated at either 25 or 37 1C. In other cases, the protoplasts were diluted into molten soft agar at 45 1C containing either LB medium and 0.5 M sucrose or M9 medium, 0.5 M sucrose, and either Leu (100 mg/ml) or Arg (100 mg/ml), depending on the nutritional requirements of the auxotrophic strains used, and then poured onto soft agar plates of the same composition.
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2.4. Protoplast fusion
3. Results
Protoplasts were fused using a modification of previously reported methods (Dai and Copley, 2004). Aliquots (0.5 ml) of protoplasts formed from each auxotroph were mixed and 1 U of DNaseI (Promega) was added to digest DNA released from lysed cells and thereby to prevent transformation resulting from uptake of DNA released from cells that had lysed during protoplast formation. After incubation at 25 1C for 10 min, the protoplasts were harvested by centrifugation at 2000g for 20 min at 4 1C and resuspended in 0.5 ml PEG buffer (SMM containing 40% v/v PEG6000, 10 mM CaCl2, and 5% v/v DMSO). After incubation at 25 1C for either 5 or 20 min, 1 ml of SMM buffer was added and the cells were harvested by centrifugation at 2000g and 4 1C for 20 min. The cells were resuspended in 0.5 ml LB medium containing 0.5 M sucrose and serial dilutions were immediately spread on soft agar LB plates containing 8 g/L agar and 0.5 M sucrose using a plastic spreader. After growth on these plates, individual colonies were patched onto plates containing M9 agar, M9 agar containing leucine (100 mg/ml), and M9 agar containing arginine (100 mg/ml). Colonies growing on each type of plate were counted after growth at 37 1C for 48 h. Colonies obtained on M9 agar plates with no amino acid supplements were patched onto M9 agar plates twice more to confirm their ability to grow without Leu or Arg. Control experiments were carried out using the above procedures but without either addition of PEG6000 or mixing of the protoplasts formed from the two auxotrophs. Additional control experiments to investigate the possibility of cross-feeding were carried out by patching the two parental strains together on M9 agar plates.
Initial experiments to optimize conditions for formation and regeneration of protoplasts were carried out with the two auxotrophic strains of E. coli that were to be used for protoplast fusion experiments. Strain W4183 is an arginine auxotroph, and Strain FU20-1 is a leucine auxotroph. Two protocols for formation of protoplasts were used. One protocol was a modification of the method used by Weiss (1976), which involves treating cells with lysozyme and EDTA. The other protocol was a further modification in which an initial treatment with EDTA was used to begin removal of the outer membrane. Subsequently, the EDTA was removed and lysozyme was added to digest the exposed peptidoglycan. Both methods gave comparable protoplast formation frequencies as judged by osmotic fragility (497%, data not shown).1 However, the results shown in Table 1 suggest that protoplasts formed by the second protocol are better able to regenerate. Thus, the second protocol was used for further experiments. Fig. 1 shows electron micrographs of cells collected during generation of protoplasts using this protocol. For most of the cells in the field, the outer membrane has peeled away from the inner membrane (see Fig. 1a) or is no longer visible (Fig. 1b). Table 1 also summarizes results of experiments designed to identify optimal conditions for protoplast regeneration. We found that protoplast regeneration is slightly more efficient at 25 1C than at 37 1C. However, exposure to molten top agar at 45 1C, a commonly used procedure for spreading protoplasts on plates, results in a 5–10-fold reduction in regeneration frequency. Further, regeneration efficiency is much more efficient on medium containing LB than on medium containing M9. The best results were achieved by spreading protoplasts on soft agar plates containing LB and 0.5 M sucrose and incubating at 25 1C. This protocol gave consistently high regeneration frequencies averaging 65% in three separate experiments (see Table 2) and was used for subsequent protoplast fusion experiments. Protoplast fusion experiments were carried out using Strain W4183 (the arginine auxotroph) and Strain FU20-1 (the leucine auxotroph). Recombination between the genomes of these two strains can lead to strains that grow on M9 medium in the absence of both arginine and leucine. Protoplasts were generated from each auxotroph by treatment with EDTA and lysozyme as described above and subjected to fusion with PEG6000 for various times. Fig. 2 shows several
2.5. Electron microscopy Samples taken during protoplast generation, fusion, and regeneration were centrifuged and fixed in 2% glutaraldehyde in 50 mM sodium cacodylate buffer, pH 7.2 at 4 1C overnight. The pellets were then washed 3 times in the same buffer, postfixed in 1% osmium tetroxide in sodium cacodylate buffer for 1 h at room temperature, and immersed in 1% uranyl acetate for 2 h. The fixed samples were dehydrated with acetone and embedded in Spurr’s epoxy resin. Thin sections (60 nm thick) were collected on formvar-coated EM grids and stained with uranyl acetate and lead citrate. Images were obtained on a Philips CM10 (FEI Co., Hillsboro, OR) transmission electron microscope operating at 80 kV with a Gatan (Pleasanton, CA) Bioscan digital camera.
1 We use the term ‘‘protoplast’’ to indicate osmotically sensitive cells that lack at least part of the outer membrane. Electron micrographs indicate that the degree of removal of the outer membrane varies among cells, and some cells retain patches of outer membrane.
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Table 1 Effect of protoplast generation and recovery conditions on protoplast regeneration frequency Protoplast generation method
Regeneration medium
Plating method
Regeneration temperature (1C)
Regeneration frequency E. coli W4183 (Arg auxotroph) (%)
E. coli FU20-1 (Leu auxotroph) (%)
(1) Lysozyme (2) EDTA
LB+0.5 M sucrose
Spreading
25
20
35
(1) EDTA (2) Lysozyme
LB+0.5 M sucrose
Spreading
25
50
75
(1) EDTA (2) Lysozyme
LB+0.5 M sucrose
Spreading
37
45
59
(1) EDTA (2) Lysozyme
LB+0.5 M sucrose
Dilution in soft agar (45 1C)
25
5
16
(1) EDTA (2) Lysozyme
M9+either Arg or Leu, as required
Dilution in soft agar (45 1C)
25
0.3
Fig. 1. Electron micrographs of cells collected during formation of protoplasts: (a) a cell for which removal of the outer membrane is in progress; and (b) a protoplast with no visible trace of outer membrane.
examples of protoplasts that have clearly resulted from fusion of two or three protoplasts. We note that some fusants have remnants of the outer membrane (Fig. 2c); thus, it is not necessary to remove the entire outer membrane in order to achieve fusion. After fusion, the protoplasts were allowed to regenerate on soft agar LB plates containing 0.5 M sucrose. Recoveries of viable cells after fusion were in the range of 5–52% (see Table 3), somewhat lower than those obtained from protoplasts that had not been subjected to fusion, but still in an acceptable range. Colonies obtained after regeneration on LB sucrose plates were patched onto plates containing M9 supplemented with either leucine or arginine or M9 with no amino acid supplements. In three separate experiments, most of the colonies obtained were still auxotrophs, and required either Leu or Arg for growth. However, 8–25% were able to grow on M9 without supplementation. However, when these colonies were repatched onto new
0.5
M9 plates, only a fraction retained the ability to grow (see Table 3). Each colony that grew on M9 plates after the third patch was streaked onto an LB plate, and five individual colonies tested for the ability to grow on M9 plates. In every case, all of the colonies retained the ability to grow on M9. Thus, the colonies obtained after the third patch were composed of stable prototrophs. The generation of prototrophs obtained in these experiments can be attributed to recombination between parental genomes in fused diploid or multiploid protoplasts rather than spontaneous reversion of mutations or transfer of genetic material by conjugation or transformation. No prototrophic colonies were obtained if the protoplasts were mixed without PEG6000, or if the individual auxotrophs were subjected to protoplasting and fusion. The experiments were carried out with F strains that are deficient in conjugation. Furthermore, the inclusion of DNaseI in the fusion medium minimizes the possibility that genetic material was transferred by uptake of DNA released from lysed cells. Finally, the possibility that growth on M9 after fusion was due to cross-feeding between the two auxotrophic strains that segregated from a heterodiploid before recombination was eliminated by experiments in which the two parental strains were patched together onto M9, but no growth was observed.
4. Discussion Tables 1 and 2 summarize the efficiencies of formation and regeneration of protoplasts of two auxotrophic strains of E. coli. Protoplast formation, as judged by osmotic fragility and direct visualization by electron microscopy, is quite efficient. Typically, protoplast formation efficiencies of 95–98% could be achieved.
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Table 2 Frequencies of protoplast formation and regeneration achieved in several experiments Expt.
Strain
Cells/ml before protoplast generationa (Z)
Viable cells/ml after protoplast generationb (X)
Viable cells/ml after lysis of protoplastsc (Y)
Protoplast formation frequency (X Y =X ) (%)
Regeneration frequency (X Y =Z) (%)
1
W4183 (Arg2 ) FU20-1 (Leu )
1.3 108 6.3 107
1.0 108 4.9 107
6.3 106 1.6 106
94 97
75 75
2
W4183 (Arg2 ) FU20-1 (Leu )
2.8 108 1.2 108
1.9 108 7.5 107
8.8 106 3.0 106
97 98
66 59
3
W4183 (Arg2 ) FU20-1 (Leu )
4.1 108 1.9 108
1.9 108 1.5 108
5.4 106 1.4 107
99 93
45 70
a
Assessed by counting colonies on LB plates before addition of lysozyme and EDTA. Assessed by counting colonies on soft agar LB plates containing 0.5 M sucrose. c Assessed by counting colonies on LB plates after dilution of protoplasts with distilled water. b
Fig. 2. Electron micrographs of several individual fusants formed by fusion of protoplasts of two auxotrophic strains of E. coli. Note in (c) that one of the two cells that have fused retains part of the outer membrane.
Protoplasts were allowed to regenerate on soft agar plates containing LB and 0.5 M sucrose. Remarkably, 34–75% of the protoplasts were able to form colonies, depending on the experimental conditions, a substantial increase in regeneration frequency compared to the reported regeneration frequencies of 0.1–1% for E. coli
protoplasts (Tsenin et al., 1978) and a moderate increase compared to the regeneration frequency of 23–28% reported for Fusobacterium varium protoplasts (Chen et al., 1986). The success of protoplast formation and regeneration is likely to be organism-specific, so the comparison with previous work using E. coli is most relevant. Our results suggest that the poor regeneration frequencies in the previously reported work with E. coli (Tsenin et al., 1978) may have been due to a combination of a less gentle method for generating protoplasts and the exposure of the fused protoplasts to molten toplayer agar at 45 1C, and possibly to the medium used for regeneration. After fusion of protoplasts from the Leu auxotroph and the Arg auxotroph and regeneration on soft agar plates containing LB and 0.5 M sucrose, a remarkably high 10–25% of the colonies are able to grow when patched onto M9 plates lacking Arg and Leu. Notably, most of the colonies that grew on the first M9 plate were unable to grow when transferred to a new M9 plate. This finding is consistent with the previous report of Tsenin et al. (1978) who observed that only about 10% of the initial prototrophic colonies obtained from fusion of two auxotrophic strains of E. coli contained actual prototrophs. These findings raise an important issue with respect to evaluation of previous reports of the efficiency of protoplast fusion, since the number of prototrophs is often assessed by the phenotype of colonies immediately after fusion, and thus the actual number of prototrophs may be overestimated. The data reported in Table 3 are consistent with the scenario depicted in Fig. 3. We expect that the initial colonies formed on soft agar containing LB medium and 0.5 M sucrose resulted from fused protoplasts containing genomic DNA from two parents. It appears that if fusion occurs between two auxotrophic parents, complementation between the two parental genomes can support growth on M9 medium even if recombination
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Table 3 Generation of prototrophs by fusion of protoplasts from two auxotrophic parental strains of E. coli Expt.
Fusion Recovery after time (min) fusion (%)
1ab 1bb 1cc 1dd
5 20 5 5
24 52 52 26
2ab 2bb
5 20
3b
20
Colonies tested
% that grow on M9+Leu
% that grow on M9+Arg
% that grow % that grow on M9 on first on M9 after patch second patch
% that grow on M9 after third patch
Overall frequency of prototroph formationa (%)
320 320 320 320
32 33 33 34
68 67 67 66
9 15 12 11
1.6 6.9 3.1 2.2
0.6 1.3 1.3 0.6
0.14 0.68 0.68 0.16
19 5
1280 896
42 52
59 49
8 8
1.6 1.7
0.4 1
0.8 0.05
22
608
43
57
25
1.2
0.2
0.04
a
Calculated by multiplying the % of colonies obtained after regeneration on LB sucrose that were capable of growth on M9 after the third patch by the fraction of protoplasts capable of regeneration after fusion. b Cells plated on LBSS immediately after fusion. c Cells plated on LBSS after 1 h of shaking at 37 1C. d Cells plated on LBSS after 1 h of standing at 37 1C.
Leu auxotroph
Arg auxotroph
complementing heterodiploid cells
(grow on M9)
recombination that fails to combine wild type alleles, followed by recombination segregation that combines wild type alleles, followed by segregation
segregation before recombination of auxotrophic markers
auxotrophs
auxotrophs
(grow on M9 for several generations)
(grow on M9 for several generations) true prototrophs (grow on M9 indefinitely)
Fig. 3. Model for the fate of heterodiploid cells obtained by fusion of protoplasts of two auxotrophic strains of E. coli.
has not occurred to combine wild type alleles into a single chromosome. During growth of the initial colonies on either LB sucrose or on M9, recombination can occur to generate true prototrophs. Alternatively, the parental genomes may segregate during cell division without recombination, or recombination may occur in such a way that wild type alleles are not combined. In the latter cases, the resulting strains will be auxotrophs, but they will retain the enzymes that were present in the biparental progenitor. In the absence of significant protein turnover, the concentration of these enzymes
will be reduced by one-half during each cell division, so that there is likely to be enough enzyme to allow synthesis of leucine or arginine for several cell generations. In such cases, colonies will appear on M9 plates, but will grow slowly and the cells will not be able to sustain growth on M9 after the enzymes responsible for the defective step have been sufficiently diluted. This model predicts that the colonies obtained after regeneration on LB sucrose and in many cases after patching on M9 will be heterogeneous. Such heterogeneity has been well-documented in previous studies of protoplast
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fusion (Hopwood and Wright, 1978; Grandjean et al., 1996; Tsenin et al., 1978; Coetzee et al., 1979). Based upon the results reported here, we suggest that the efficiency of protoplast fusion is critically dependent upon both technical and organismic factors. It is important to generate protoplasts and to allow regeneration after fusion under gentle conditions so that irreversible damage to the cells does not occur. We have observed fusion between cells that still retain some outer membrane and thus would technically be considered spheroplasts rather than true protoplasts (see Fig. 2c). Thus, previously raised concerns about the difficulty of completely removing the outer membrane of Gramnegative organisms may not be justified (Hopwood, 1981). Other factors include the ability of two genomes in a heterodiploid fusion product to complement each other, the rate of recombination between genomes, and the rate of segregation of extra chromosomes into individual haploid cells. Recombination frequencies are known to depend upon the number and location of the markers used (Schaeffer et al., 1976; Coetzee et al., 1979) Some of these factors may be organism-specific. For example, fusion of B. subtilis protoplasts has been reported to result in non-complementing heterodiploids in which one chromosome is inactivated (Hotchkiss and Gabor, 1980; Grandjean et al., 1996), while our results suggest that fusion of E. coli protoplasts results in complementing heterodiploids. Furthermore, recombination frequencies in fused protoplasts of S. coelicolor range from 10% to 17% (Hopwood and Wright, 1978), but appear to be very much lower in fused protoplasts of B. subtilis (Grandjean et al., 1996). We were able to reproducibly generate prototrophs at frequencies in the range of 0.05–0.7% based upon the number of protoplasts subjected to fusion, and 0.2–1.3% of the protoplasts that were capable of regenerating on solid medium. Thus, our procedures were markedly more effective than those reported by Tsenin et al., who reported recombination frequencies of only 10 6 using auxotrophic strains of E. coli (Tsenin et al., 1978) and by Coetzee et al. (1979) who achieved recombination frequencies of 3.5 10 6 based upon the number of protoplasts subjected to fusion using auxotrophic strains of P. alcalifaciens. We attribute these differences in large part to the improved frequency of protoplast regeneration in our experiments. Improved regeneration frequency is a particularly key factor with respect to genome shuffling, which relies on recursive protoplast fusion, and thus the overall recovery depends in a multiplicative way on the regeneration frequency after each step of protoplast fusion. Differences in recombination frequencies in fused protoplasts and in the efficiency of segregation of prototrophs may also be a factor. In the experiments with P. alcalifaciens, prototrophs were generated at a frequency of 3 10 5 per regenerating protoplast (Coetzee et al., 1979), while
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we obtained frequencies of 0.5–1.7 10 2 per regenerating protoplast. In a recent report of successful genome shuffling in Streptomyces coelicolor, 8.4% of the cells obtained after fusion of four parental strains had recombined two markers. In our experiments, 0.2–1.3% of the cells obtained after fusion of two parental strains had recombined two markers. Thus, the efficiency of recombination during protoplast fusion in E. coli is only marginally lower than that obtained in the most successful reports using Gram-positive organisms. Given the improvement in recovery of cells after fusion of protoplasts obtained using the methods described here, we believe that genome shuffling will be useful for efforts to evolve improved strains in E. coli and likely other Gram-negative bacteria, as well.
Acknowledgements We thank Dr. Thomas Giddings for preparing the electron micrographs shown in Figs. 1 and 2. This work was supported by NIH R21 AI055773-01.
References Chen, W., Ohmiya, K., Shimizu, S., 1986. Protoplast formation and regeneration of dehydrovanillin-degrading strains of Fusobacterium varium and Enterococcus faecium. Appl. Env. Microbiol. 52, 612–616. Coetzee, J.N., Sirgel, F.A., Lecatsas, G., 1979. Genetic recombination in fused spheroplasts of Providence alcalifaciens. J. Gen. Microbiol. 114, 313–322. Dai, M.-H., Copley, S.D., 2004. Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. Appl. Environ. Microbiol. 70, 2391–2397. Grandjean, V., Hauck, Y., Le De´rout, J., Hirschbein, L., 1996. Noncomplementing diploids from Bacillus subtilis protoplast fusion: relationship between maintenance of chromosomal inactivation and segregation capacity. Genetics 144, 871–881. Hanson, K.G., Desai, A.J., 1996. Intergeneric protoplast fusion between Acinetobacter sp. A3 and Pseudomonas putida DP99 for enhanced hydrocarbon degradation. Biotechnol. Lett. 18, 1369–1374. Hopwood, D.A., 1981. Genetic studies with bacterial protoplasts. Annu. Rev. Microbiol. 35, 237–272. Hopwood, D.A., Wright, H.M., 1978. Bacterial protoplast fusion: recombination in fused protoplasts of Streptomyces coelicolor. Mol. Gen. Genet. 162, 307–317. Hotchkiss, R.D., Gabor, M.H., 1980. Biparental products of bacterial protoplast fusion showing unequal parental chromosome expression. Proc. Natl. Acad. Sci. USA 77, 3553–3557. Patnaik, R., Louie, S., Gavrilovic, V., Perry, K., Stemmer, W.P.C., Ryan, C.M., del Cardayre´, S., 2002. Genome shuffling of Lactobacillus for improved acid tolerance. Nature Biotechnol. 20, 707–712. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Metabolic Engineering 7 (2005) 45–52 www.elsevier.com/locate/ymben
Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli MingHua Daia, Sara Ziesmanb, Thomas Ratcliffeb, Ryan T. Gillb, Shelley D. Copleya, a
Department of Molecular, Cellular, and Developmental Biology and Cooperative Institute for Research in Environmental Sciences, University of Colorado at Boulder, Boulder, CO 80309, USA b Department of Chemical and Biological Engineering, University of Colorado at Boulder, Boulder, CO 80309, USA Received 12 May 2004; accepted 13 September 2004 Available online 10 November 2004
Abstract Protoplast fusion has been used to combine genes from different organisms to create strains with desired properties. A recently developed variant on this approach, genome shuffling, involves generation of a genetically heterogeneous population of a single organism, followed by recursive protoplast fusion to allow recombination of mutations within the fused protoplasts. These are powerful techniques for engineering of microbial strains for desirable industrial properties. However, there is a prevailing opinion that it will be difficult to use these methods for engineering of Gram-negative bacteria because the outer membrane makes protoplast fusion more difficult. Here we describe the successful use of protoplast fusion in Escherichia coli. Using two auxotrophic strains of E. coli, we obtained prototrophic strains by recombination in fused protoplasts at frequencies of 0.05–0.7% based on the number of protoplasts subjected to fusion. This frequency is three–four orders of magnitude better than those previously reported for recombination in fused protoplasts of Gram-negative bacteria such as E. coli and Providencia alcalifaciens. r 2004 Elsevier Inc. All rights reserved.
1. Introduction Classical methods for improvement of microbial strains have relied upon either mutagenesis followed by selection for improved properties, or manipulation of specific genes known to play an important role in the desired phenotype. The first strategy is time-consuming, requiring many generations of mutation and selection to allow accumulation of multiple beneficial mutations in a single strain. The efficacy of the second strategy is limited by the ability to predict which mutations will improve a particular phenotype. Thus, it is not possible to take advantage of mutations in genes that are not obviously related to the phenotype of interest but may nevertheless improve microbial fitness or performance under a particular set of conditions. Genome shuffling is a recently introduced method that is much more efficient Corresponding author.
E-mail address: [email protected] (S.D. Copley). 1096-7176/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymben.2004.09.002
for evolution of strains of microbes with desirable phenotypes. It involves generation of a heterogeneous population of mutants by treatment with a chemical mutagen, followed by recursive fusion of protoplasts to allow recombination. This procedure allows rapid evolution of strains with multiple beneficial mutations. For example, recent reports have described the use of genome shuffling to improve production of tylosin by Streptomyces fradiae (Zhang et al., 2002), acid tolerance in Lactobacillus (Patnaik et al., 2002), and degradation of pentachlorophenol in Sphingobium chlorophenolicum (Dai and Copley, 2004). Other reports have described successful use of protoplast fusion (without the initial mutagenesis step) to combine metabolic capabilities of two different organisms. For example, protoplasts of Acinetobacter sp. A3 and Pseudomonas putida DP99 have been fused to generate strains with enhanced abilities to degrade hydrocarbons (Hanson and Desai, 1996), and protoplasts of Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15 have been fused to
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generate strains with an enhanced ability to produce sorbitol under fermentation conditions (Wei et al., 2001). The efficiency of genome shuffling is a function of the efficiencies of generation and fusion of protoplasts, of recovery of protoplasts after fusion, of recombination between heterogeneous chromosomes in the diploid or multiploid cells obtained after fusion, and of the eventual segregation of true prototrophs containing only one genome. Protoplasts are typically generated by treatment of cells with lysozyme to digest the peptidoglycan surrounding the inner membrane (Weiss, 1976; Hopwood, 1981). This process is quite efficient in Grampositive bacteria, but less so in Gram-negative bacteria because the outer membrane impedes access of lysozyme to the peptidoglycan layer. Indeed, it is a commonly held notion that protoplast fusion in Gram-negative organisms is too challenging for use as a strain engineering tool. This skepticism appears to be based on a small number of early studies reporting limited success in fusion of protoplasts from Gram-negative organisms. For example, Tsenin et al. (1978) reported generation of prototrophic colonies from fusion of protoplasts of two auxotrophs of Escherichia coli K12 at a frequency of only 1 10 5, and only 10% of these colonies appeared to be true prototrophs. Coetzee et al. (1979) reported generation of prototrophic colonies at frequencies ranging from 1.7 10 7 to 3.8 10 6 per protoplast originally present using auxotrophs of Providencia alcalifaciens strain P29. We have recently reported the use of genome shuffling in the Gram-negative bacterium S. chlorophenolicum to generate strains with an enhanced ability to degrade the toxic pesticide pentachlorophenol (Dai and Copley, 2004) and have begun to explore the use of genome shuffling in E. coli. Here we provide data demonstrating that the efficiency of recombination in fused E. coli protoplasts is lower than that achieved using Grampositive organisms, but is nevertheless high enough for practical applications. We have used electron microscopy to visualize the formation and fusion of protoplasts during the procedure, and have quantified the generation of prototrophic strains by recombination of parental genomes in fused protoplasts generated from two auxotrophic parental strains. These data provide encouragement for efforts to utilize genome shuffling in Gram-negative bacteria.
2. Materials and methods 2.1. Bacterial strains and growth conditions E. coli W4183: argG78, rpsL257, (strR), F , and E. coli FU20-1: leuB6(Am), phoA1, rpsL114(strR), malT1(lR), F were obtained from the E. coli Genetic
Stock Center (http://cgsc.biology.yale.edu). Cells were grown in either LB or M9 medium (Sambrook and Russell, 2001) supplemented with either arginine or leucine (100 mg/ml). All media contained streptomycin (10 mg/ml).
2.2. Protoplast formation Protoplasts were formed by modification of a previously reported method (Weiss, 1976). Each auxotroph was grown in 30 ml of LB medium containing streptomycin (10 mg/ml) until an OD550 of about 1.0 was reached. The cells were harvested by centrifugation at 3000g and 4 1C for 10 min, washed three times with 0.01 M Tris-HCl (pH 8.0), and resuspended in 30 ml 0.01 M Tris-HCl (pH 8.0) containing 0.5 M sucrose. Potassium ethylenediaminetetraacetate (EDTA, 0.1 M, pH 8.0) was added slowly over a period of 20 min to a final concentration of 0.01 M. The cells were shaken at 100 rpm at 37 1C for an additional 20 min to begin removal of the outer membrane. The cells were harvested by centrifugation at 3000g and 4 1C for 10 min and washed twice with SMM buffer (0.5 M sucrose, 20 mM sodium maleate, 20 mM MgCl2, pH 6.5). The cells were resuspended in 30 ml SMM buffer containing lysozyme (1 mg/ml). The cells were shaken at 37 1C at 100 rpm for 1 h to allow digestion of the peptidoglycan layer. For one experiment, cells were suspended in SMM buffer containing lysozyme (1 mg/ ml), and the cells were shaken at 100 rpm at 37 1C for 1 h. Subsequently, EDTA was added slowly to a final concentration of 0.01 M and the cells were shaken at 37 1C for 20 min. The efficiency of protoplast formation was judged by subjecting an aliquot of the cells to dilution in distilled water to lyse protoplasts, followed by plating of serial dilutions on LB agar to quantify colonies resulting from cells that had not formed protoplasts.
2.3. Protoplast regeneration Several conditions for regeneration of protoplasts were explored. In some cases, protoplasts were resuspended in LB medium containing 0.5 M sucrose and spread onto soft agar plates containing LB medium and 0.5 M sucrose with autoclaved plastic spreaders. The plates were subsequently incubated at either 25 or 37 1C. In other cases, the protoplasts were diluted into molten soft agar at 45 1C containing either LB medium and 0.5 M sucrose or M9 medium, 0.5 M sucrose, and either Leu (100 mg/ml) or Arg (100 mg/ml), depending on the nutritional requirements of the auxotrophic strains used, and then poured onto soft agar plates of the same composition.
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2.4. Protoplast fusion
3. Results
Protoplasts were fused using a modification of previously reported methods (Dai and Copley, 2004). Aliquots (0.5 ml) of protoplasts formed from each auxotroph were mixed and 1 U of DNaseI (Promega) was added to digest DNA released from lysed cells and thereby to prevent transformation resulting from uptake of DNA released from cells that had lysed during protoplast formation. After incubation at 25 1C for 10 min, the protoplasts were harvested by centrifugation at 2000g for 20 min at 4 1C and resuspended in 0.5 ml PEG buffer (SMM containing 40% v/v PEG6000, 10 mM CaCl2, and 5% v/v DMSO). After incubation at 25 1C for either 5 or 20 min, 1 ml of SMM buffer was added and the cells were harvested by centrifugation at 2000g and 4 1C for 20 min. The cells were resuspended in 0.5 ml LB medium containing 0.5 M sucrose and serial dilutions were immediately spread on soft agar LB plates containing 8 g/L agar and 0.5 M sucrose using a plastic spreader. After growth on these plates, individual colonies were patched onto plates containing M9 agar, M9 agar containing leucine (100 mg/ml), and M9 agar containing arginine (100 mg/ml). Colonies growing on each type of plate were counted after growth at 37 1C for 48 h. Colonies obtained on M9 agar plates with no amino acid supplements were patched onto M9 agar plates twice more to confirm their ability to grow without Leu or Arg. Control experiments were carried out using the above procedures but without either addition of PEG6000 or mixing of the protoplasts formed from the two auxotrophs. Additional control experiments to investigate the possibility of cross-feeding were carried out by patching the two parental strains together on M9 agar plates.
Initial experiments to optimize conditions for formation and regeneration of protoplasts were carried out with the two auxotrophic strains of E. coli that were to be used for protoplast fusion experiments. Strain W4183 is an arginine auxotroph, and Strain FU20-1 is a leucine auxotroph. Two protocols for formation of protoplasts were used. One protocol was a modification of the method used by Weiss (1976), which involves treating cells with lysozyme and EDTA. The other protocol was a further modification in which an initial treatment with EDTA was used to begin removal of the outer membrane. Subsequently, the EDTA was removed and lysozyme was added to digest the exposed peptidoglycan. Both methods gave comparable protoplast formation frequencies as judged by osmotic fragility (497%, data not shown).1 However, the results shown in Table 1 suggest that protoplasts formed by the second protocol are better able to regenerate. Thus, the second protocol was used for further experiments. Fig. 1 shows electron micrographs of cells collected during generation of protoplasts using this protocol. For most of the cells in the field, the outer membrane has peeled away from the inner membrane (see Fig. 1a) or is no longer visible (Fig. 1b). Table 1 also summarizes results of experiments designed to identify optimal conditions for protoplast regeneration. We found that protoplast regeneration is slightly more efficient at 25 1C than at 37 1C. However, exposure to molten top agar at 45 1C, a commonly used procedure for spreading protoplasts on plates, results in a 5–10-fold reduction in regeneration frequency. Further, regeneration efficiency is much more efficient on medium containing LB than on medium containing M9. The best results were achieved by spreading protoplasts on soft agar plates containing LB and 0.5 M sucrose and incubating at 25 1C. This protocol gave consistently high regeneration frequencies averaging 65% in three separate experiments (see Table 2) and was used for subsequent protoplast fusion experiments. Protoplast fusion experiments were carried out using Strain W4183 (the arginine auxotroph) and Strain FU20-1 (the leucine auxotroph). Recombination between the genomes of these two strains can lead to strains that grow on M9 medium in the absence of both arginine and leucine. Protoplasts were generated from each auxotroph by treatment with EDTA and lysozyme as described above and subjected to fusion with PEG6000 for various times. Fig. 2 shows several
2.5. Electron microscopy Samples taken during protoplast generation, fusion, and regeneration were centrifuged and fixed in 2% glutaraldehyde in 50 mM sodium cacodylate buffer, pH 7.2 at 4 1C overnight. The pellets were then washed 3 times in the same buffer, postfixed in 1% osmium tetroxide in sodium cacodylate buffer for 1 h at room temperature, and immersed in 1% uranyl acetate for 2 h. The fixed samples were dehydrated with acetone and embedded in Spurr’s epoxy resin. Thin sections (60 nm thick) were collected on formvar-coated EM grids and stained with uranyl acetate and lead citrate. Images were obtained on a Philips CM10 (FEI Co., Hillsboro, OR) transmission electron microscope operating at 80 kV with a Gatan (Pleasanton, CA) Bioscan digital camera.
1 We use the term ‘‘protoplast’’ to indicate osmotically sensitive cells that lack at least part of the outer membrane. Electron micrographs indicate that the degree of removal of the outer membrane varies among cells, and some cells retain patches of outer membrane.
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Table 1 Effect of protoplast generation and recovery conditions on protoplast regeneration frequency Protoplast generation method
Regeneration medium
Plating method
Regeneration temperature (1C)
Regeneration frequency E. coli W4183 (Arg auxotroph) (%)
E. coli FU20-1 (Leu auxotroph) (%)
(1) Lysozyme (2) EDTA
LB+0.5 M sucrose
Spreading
25
20
35
(1) EDTA (2) Lysozyme
LB+0.5 M sucrose
Spreading
25
50
75
(1) EDTA (2) Lysozyme
LB+0.5 M sucrose
Spreading
37
45
59
(1) EDTA (2) Lysozyme
LB+0.5 M sucrose
Dilution in soft agar (45 1C)
25
5
16
(1) EDTA (2) Lysozyme
M9+either Arg or Leu, as required
Dilution in soft agar (45 1C)
25
0.3
Fig. 1. Electron micrographs of cells collected during formation of protoplasts: (a) a cell for which removal of the outer membrane is in progress; and (b) a protoplast with no visible trace of outer membrane.
examples of protoplasts that have clearly resulted from fusion of two or three protoplasts. We note that some fusants have remnants of the outer membrane (Fig. 2c); thus, it is not necessary to remove the entire outer membrane in order to achieve fusion. After fusion, the protoplasts were allowed to regenerate on soft agar LB plates containing 0.5 M sucrose. Recoveries of viable cells after fusion were in the range of 5–52% (see Table 3), somewhat lower than those obtained from protoplasts that had not been subjected to fusion, but still in an acceptable range. Colonies obtained after regeneration on LB sucrose plates were patched onto plates containing M9 supplemented with either leucine or arginine or M9 with no amino acid supplements. In three separate experiments, most of the colonies obtained were still auxotrophs, and required either Leu or Arg for growth. However, 8–25% were able to grow on M9 without supplementation. However, when these colonies were repatched onto new
0.5
M9 plates, only a fraction retained the ability to grow (see Table 3). Each colony that grew on M9 plates after the third patch was streaked onto an LB plate, and five individual colonies tested for the ability to grow on M9 plates. In every case, all of the colonies retained the ability to grow on M9. Thus, the colonies obtained after the third patch were composed of stable prototrophs. The generation of prototrophs obtained in these experiments can be attributed to recombination between parental genomes in fused diploid or multiploid protoplasts rather than spontaneous reversion of mutations or transfer of genetic material by conjugation or transformation. No prototrophic colonies were obtained if the protoplasts were mixed without PEG6000, or if the individual auxotrophs were subjected to protoplasting and fusion. The experiments were carried out with F strains that are deficient in conjugation. Furthermore, the inclusion of DNaseI in the fusion medium minimizes the possibility that genetic material was transferred by uptake of DNA released from lysed cells. Finally, the possibility that growth on M9 after fusion was due to cross-feeding between the two auxotrophic strains that segregated from a heterodiploid before recombination was eliminated by experiments in which the two parental strains were patched together onto M9, but no growth was observed.
4. Discussion Tables 1 and 2 summarize the efficiencies of formation and regeneration of protoplasts of two auxotrophic strains of E. coli. Protoplast formation, as judged by osmotic fragility and direct visualization by electron microscopy, is quite efficient. Typically, protoplast formation efficiencies of 95–98% could be achieved.
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Table 2 Frequencies of protoplast formation and regeneration achieved in several experiments Expt.
Strain
Cells/ml before protoplast generationa (Z)
Viable cells/ml after protoplast generationb (X)
Viable cells/ml after lysis of protoplastsc (Y)
Protoplast formation frequency (X Y =X ) (%)
Regeneration frequency (X Y =Z) (%)
1
W4183 (Arg2 ) FU20-1 (Leu )
1.3 108 6.3 107
1.0 108 4.9 107
6.3 106 1.6 106
94 97
75 75
2
W4183 (Arg2 ) FU20-1 (Leu )
2.8 108 1.2 108
1.9 108 7.5 107
8.8 106 3.0 106
97 98
66 59
3
W4183 (Arg2 ) FU20-1 (Leu )
4.1 108 1.9 108
1.9 108 1.5 108
5.4 106 1.4 107
99 93
45 70
a
Assessed by counting colonies on LB plates before addition of lysozyme and EDTA. Assessed by counting colonies on soft agar LB plates containing 0.5 M sucrose. c Assessed by counting colonies on LB plates after dilution of protoplasts with distilled water. b
Fig. 2. Electron micrographs of several individual fusants formed by fusion of protoplasts of two auxotrophic strains of E. coli. Note in (c) that one of the two cells that have fused retains part of the outer membrane.
Protoplasts were allowed to regenerate on soft agar plates containing LB and 0.5 M sucrose. Remarkably, 34–75% of the protoplasts were able to form colonies, depending on the experimental conditions, a substantial increase in regeneration frequency compared to the reported regeneration frequencies of 0.1–1% for E. coli
protoplasts (Tsenin et al., 1978) and a moderate increase compared to the regeneration frequency of 23–28% reported for Fusobacterium varium protoplasts (Chen et al., 1986). The success of protoplast formation and regeneration is likely to be organism-specific, so the comparison with previous work using E. coli is most relevant. Our results suggest that the poor regeneration frequencies in the previously reported work with E. coli (Tsenin et al., 1978) may have been due to a combination of a less gentle method for generating protoplasts and the exposure of the fused protoplasts to molten toplayer agar at 45 1C, and possibly to the medium used for regeneration. After fusion of protoplasts from the Leu auxotroph and the Arg auxotroph and regeneration on soft agar plates containing LB and 0.5 M sucrose, a remarkably high 10–25% of the colonies are able to grow when patched onto M9 plates lacking Arg and Leu. Notably, most of the colonies that grew on the first M9 plate were unable to grow when transferred to a new M9 plate. This finding is consistent with the previous report of Tsenin et al. (1978) who observed that only about 10% of the initial prototrophic colonies obtained from fusion of two auxotrophic strains of E. coli contained actual prototrophs. These findings raise an important issue with respect to evaluation of previous reports of the efficiency of protoplast fusion, since the number of prototrophs is often assessed by the phenotype of colonies immediately after fusion, and thus the actual number of prototrophs may be overestimated. The data reported in Table 3 are consistent with the scenario depicted in Fig. 3. We expect that the initial colonies formed on soft agar containing LB medium and 0.5 M sucrose resulted from fused protoplasts containing genomic DNA from two parents. It appears that if fusion occurs between two auxotrophic parents, complementation between the two parental genomes can support growth on M9 medium even if recombination
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Table 3 Generation of prototrophs by fusion of protoplasts from two auxotrophic parental strains of E. coli Expt.
Fusion Recovery after time (min) fusion (%)
1ab 1bb 1cc 1dd
5 20 5 5
24 52 52 26
2ab 2bb
5 20
3b
20
Colonies tested
% that grow on M9+Leu
% that grow on M9+Arg
% that grow % that grow on M9 on first on M9 after patch second patch
% that grow on M9 after third patch
Overall frequency of prototroph formationa (%)
320 320 320 320
32 33 33 34
68 67 67 66
9 15 12 11
1.6 6.9 3.1 2.2
0.6 1.3 1.3 0.6
0.14 0.68 0.68 0.16
19 5
1280 896
42 52
59 49
8 8
1.6 1.7
0.4 1
0.8 0.05
22
608
43
57
25
1.2
0.2
0.04
a
Calculated by multiplying the % of colonies obtained after regeneration on LB sucrose that were capable of growth on M9 after the third patch by the fraction of protoplasts capable of regeneration after fusion. b Cells plated on LBSS immediately after fusion. c Cells plated on LBSS after 1 h of shaking at 37 1C. d Cells plated on LBSS after 1 h of standing at 37 1C.
Leu auxotroph
Arg auxotroph
complementing heterodiploid cells
(grow on M9)
recombination that fails to combine wild type alleles, followed by recombination segregation that combines wild type alleles, followed by segregation
segregation before recombination of auxotrophic markers
auxotrophs
auxotrophs
(grow on M9 for several generations)
(grow on M9 for several generations) true prototrophs (grow on M9 indefinitely)
Fig. 3. Model for the fate of heterodiploid cells obtained by fusion of protoplasts of two auxotrophic strains of E. coli.
has not occurred to combine wild type alleles into a single chromosome. During growth of the initial colonies on either LB sucrose or on M9, recombination can occur to generate true prototrophs. Alternatively, the parental genomes may segregate during cell division without recombination, or recombination may occur in such a way that wild type alleles are not combined. In the latter cases, the resulting strains will be auxotrophs, but they will retain the enzymes that were present in the biparental progenitor. In the absence of significant protein turnover, the concentration of these enzymes
will be reduced by one-half during each cell division, so that there is likely to be enough enzyme to allow synthesis of leucine or arginine for several cell generations. In such cases, colonies will appear on M9 plates, but will grow slowly and the cells will not be able to sustain growth on M9 after the enzymes responsible for the defective step have been sufficiently diluted. This model predicts that the colonies obtained after regeneration on LB sucrose and in many cases after patching on M9 will be heterogeneous. Such heterogeneity has been well-documented in previous studies of protoplast
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fusion (Hopwood and Wright, 1978; Grandjean et al., 1996; Tsenin et al., 1978; Coetzee et al., 1979). Based upon the results reported here, we suggest that the efficiency of protoplast fusion is critically dependent upon both technical and organismic factors. It is important to generate protoplasts and to allow regeneration after fusion under gentle conditions so that irreversible damage to the cells does not occur. We have observed fusion between cells that still retain some outer membrane and thus would technically be considered spheroplasts rather than true protoplasts (see Fig. 2c). Thus, previously raised concerns about the difficulty of completely removing the outer membrane of Gramnegative organisms may not be justified (Hopwood, 1981). Other factors include the ability of two genomes in a heterodiploid fusion product to complement each other, the rate of recombination between genomes, and the rate of segregation of extra chromosomes into individual haploid cells. Recombination frequencies are known to depend upon the number and location of the markers used (Schaeffer et al., 1976; Coetzee et al., 1979) Some of these factors may be organism-specific. For example, fusion of B. subtilis protoplasts has been reported to result in non-complementing heterodiploids in which one chromosome is inactivated (Hotchkiss and Gabor, 1980; Grandjean et al., 1996), while our results suggest that fusion of E. coli protoplasts results in complementing heterodiploids. Furthermore, recombination frequencies in fused protoplasts of S. coelicolor range from 10% to 17% (Hopwood and Wright, 1978), but appear to be very much lower in fused protoplasts of B. subtilis (Grandjean et al., 1996). We were able to reproducibly generate prototrophs at frequencies in the range of 0.05–0.7% based upon the number of protoplasts subjected to fusion, and 0.2–1.3% of the protoplasts that were capable of regenerating on solid medium. Thus, our procedures were markedly more effective than those reported by Tsenin et al., who reported recombination frequencies of only 10 6 using auxotrophic strains of E. coli (Tsenin et al., 1978) and by Coetzee et al. (1979) who achieved recombination frequencies of 3.5 10 6 based upon the number of protoplasts subjected to fusion using auxotrophic strains of P. alcalifaciens. We attribute these differences in large part to the improved frequency of protoplast regeneration in our experiments. Improved regeneration frequency is a particularly key factor with respect to genome shuffling, which relies on recursive protoplast fusion, and thus the overall recovery depends in a multiplicative way on the regeneration frequency after each step of protoplast fusion. Differences in recombination frequencies in fused protoplasts and in the efficiency of segregation of prototrophs may also be a factor. In the experiments with P. alcalifaciens, prototrophs were generated at a frequency of 3 10 5 per regenerating protoplast (Coetzee et al., 1979), while
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we obtained frequencies of 0.5–1.7 10 2 per regenerating protoplast. In a recent report of successful genome shuffling in Streptomyces coelicolor, 8.4% of the cells obtained after fusion of four parental strains had recombined two markers. In our experiments, 0.2–1.3% of the cells obtained after fusion of two parental strains had recombined two markers. Thus, the efficiency of recombination during protoplast fusion in E. coli is only marginally lower than that obtained in the most successful reports using Gram-positive organisms. Given the improvement in recovery of cells after fusion of protoplasts obtained using the methods described here, we believe that genome shuffling will be useful for efforts to evolve improved strains in E. coli and likely other Gram-negative bacteria, as well.
Acknowledgements We thank Dr. Thomas Giddings for preparing the electron micrographs shown in Figs. 1 and 2. This work was supported by NIH R21 AI055773-01.
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