Vol. 57, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1991, p. 1516-15220099-2240/91/051516-07$02.00/OCopyright C 1991, American Society for Microbiology

Polyethylene Glycol-Induced Internalization of Bacteria into FungalProtoplasts: Electron Microscopic Study and Optimization of

Experimental ConditionsISABEL GUERRA-TSCHUSCHKE,* INES MARTIN, AND M. TERESA GONZALEZ

Departamento de Microbiologia, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain

Received 26 October 1990/Accepted 24 February 1991

We studied the mechanism of internalization of Escherichia coli into Saccharomyces cerevisiae induced bypolyethylene glycol (PEG) and optimized the experimental conditions. Transmission electron microscopestudies revealed that the principal factor involved in the internalization was the degree of cell aggregationattained. Internalization occurred mainly by an endocytosis-like mechanism and took place during theelimination of PEG. The optimum conditions were to treat a mixed pellet of both microorganisms with 15%PEG and then gradually dilute the polymer. The same conditions were applied to E. coli and Aspergillusnidulans, with similar results.

It has been shown that when animal cells, plant proto-plasts, and fungal protoplasts are treated with polyethyleneglycol (PEG), there is a tendency for the cells to fusetogether (1, 6, 15). Nevertheless, when participants of sig-nificantly different sizes (e.g., bacteria or organelles versusfungal protoplasts, plant protoplasts, or animal cells) aretreated with PEG, the larger participant readily internalizesthe smaller one (3, 4, 13, 18, 23, 25).

In recent years, various researchers have used this tech-nique with two different aims. Some authors have attempted,with some success, to deliver foreign genes into eukaryoticcells via bacterial protoplasts harboring cloned genes (12, 14,17, 20). Others, however, have used this technique withdifferent eukaryotic-prokaryotic combinations in an attemptto establish directly a viable endosymbiotic relationship withthe ability to fix nitrogen, but these attempts have beenlargely in vain (2, 8-11, 19, 24). Nevertheless, it seems thatPEG treatment may be a useful tool for reproducing the earlystages of natural endosymbiotic relationships, skipping theentry and recognition steps, and allowing the possibility ofdetermining the response of a eukaryotic host upon the entryof a foreign microorganism.The major problem in carrying out such a study is to

achieve the largest population possible of participants con-taining a prokaryote, so that they can be easily detected andare sufficient in number to provide a statistically relevantresult. With this end in mind, we investigated the variousfactors involved in the internalization process in a model thatuses Escherichia coli cells and Saccharomyces cerevisiaeprotoplasts. We used these two participants because theyare the best known prokaryotic and eukaryotic organismsand because many strains are available for further studies.For a start, we tested the method proposed by Yamada andSakaguchi (26) for S. cerevisiae-Azotobacter vinelandii. Wealso carried out similar experiments with Aspergillus nidu-lans-E. coli to test our results and to determine whether thesize of the host protoplasts has any effect on the process.

Finally, using transmission electron microscopy (TEM),we tried to reach a conclusion concerning the nature of theinternalization process, about which there is still no concen-

* Corresponding author.

sus of opinion. Hasezawa et al. (13), for example, suggestedan endocytosis-like mechanism, while Ferenczy (5) wasinclined toward the idea that the smaller organism is trappedwithin the aggregates of the protoplasts during a process offusion. Yamada and Sakaguchi (26) suggested that a combi-nation of both processes is involved.

MATERIALS AND METHODS

Organisms and culture conditions. S. cerevisiae 3.2 (aAde- Ura- His-) was from our own laboratory collection.Cells were grown at 28°C in shaken CM medium (1%glucose, 0.5% yeast extract, 0.5% malt extract). A. nidulans1056 (Ade- PABA-), with yellowish conidia, was obtainedfrom L. Ferenczy. The slants were kept in CM medium. Forprotoplast isolation, cells were grown in MM medium [2%glucose, 0.3% (NH4)2SO4, 0.2% KH2PO4, 0.1% MgSO4,0.1% Wickerham's vitamin solution, 2 pLg of adenine per ml].E. coli JC5466(pRD1) (Trp- recA56 lacAX74) was obtainedfrom N. Willets. Cells were grown in LB medium (1%tryptone, 0.5% yeast extract, 0.5% NaCI).

Isolation of fungal protoplasts. S. cerevisiae protoplastswere obtained by treatment with "protoplast-forming en-zyme" (Boehringer Mannheim) by the method of Ferenczyand Maraz (7). A. nidulans protoplasts were obtained withthe same enzyme as that described by Kevei and Peberdy(16).

Introduction of bacterial cells into fungal protoplasts. Thestarting procedure, basically that described by Yamada andSakaguchi (26), was as follows. One milliliter of Tris-HCl(pH 8) containing 60 mM CaCl2 (TC) and 30% (wt/vol) PEG4000 (Merck) was added to a 1-ml mixture of S. cerevisiaeprotoplasts (108 cells per ml) and bacterial cells (1019 cellsper ml), and the mixture was incubated at 25 to 30°C for 30min. The cells were collected by centrifugation, washedthree times with TC containing 1 M sorbitol (TCS), andresuspended in 0.8 M sorbitol. With A. nidulans protoplasts,0.8 M KCI instead of sorbitol was used as the osmoticstabilizer.

Viability of fungal protoplasts. The viability of fungalprotoplasts after the induction of internalization was deter-mined by measuring their regeneration levels. Appropriatedilutions of fungal protoplast-bacterial cell mixtures after

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*~~~~~FIG. 1. Formation of aggregates between E. coli cells (B) and S.

cerevisiae protoplasts (P) by the method of Yamada and Sakaguchi(26).

PEG treatment were inoculated into 5 to 7 ml of 2% agar in0.8 M sorbitol kept at 45°C. They were then overlaid on

plates containing CM medium osmotically stabilized with 0.8M sorbitol. PEG-treated protoplasts without bacteria were

used as a control.DAPI staining and observation by fluorescence microscopy.

E. coli cells were grown in LB medium containing 1 ,ug ofDAPI (4',6-diamidino-2-phenylindole) per ml. The cells werecollected by centrifugation, washed several times, andmixed with protoplasts to induce the internalization of thestained bacteria. Finally, they were fixed with 2% glutaral-dehyde solution for 10 min and washed two or three timeswith distilled water (no osmotic preservatives were neces-

sary, as the protoplasts were fixed). The protoplasts were

examined under a Leitz microscope with epifluorescenceillumination.TEM. At different stages of internalization, samples were

taken and fixed overnight with 2% acrolein-4% glutaralde-hyde-4% formaldehyde in phosphate buffer (0.5 M, pH 6.5)containing 5 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and MgCl2. After beingwashed with phosphate buffer (pH 6.5), they were postfixedin osmium tetroxide for 1 h. They were then dehydrated inan acetone series and embedded in Spurr's epoxy resin (21).Ultrathin sections were stained with uranyl acetate-leadcitrate. The grids were examined under a Zeiss 902 electronmicroscope.

RESULTS

PEG concentration and yeast/bacterium ratio. At the TCSwashing stage, some small aggregations of protoplasts andbacteria were observable by TEM (Fig. 1), and occasionallysome protoplasts containing a bacterium were found. No

internalization could be seen with optical fluorescence mi-croscopy and DAPI-stained bacteria.

In subsequent experiments, the PEG solution was addeddirectly to the pellets to enhance the formation of mixedaggregates. We assayed 15 and 30% concentrations of PEGon pellets containing yeast/bacterium ratios of 1:10, 1:100,and 1:1,000. TEM studies of samples from the six possibleconditions at the TCS washing stage showed that internal-ization occurred only with 15% PEG, even at a yeast/bacterium ratio of 1:10. At 1:100, both the number ofprotoplasts containing a bacterial cell and the number ofbacteria lodged in a single protoplast increased significantly,some protoplasts containing up to three prokaryotes (Fig.2a). At 1:1,000, however, the rate of internalization droppedconsiderably.

Optical fluorescence microscopy studies were unsatisfac-tory. When 15% PEG and a yeast/bacterium ratio of 1:100were used, a few protoplasts which seemed to harborbacteria were observed, but it was difficult to decide at theoptical microscope resolution whether the bacteria were infact internalized or merely adhering to the cell membrane.Mixed yeast-bacterium aggregates, in cases in which it wasimpossible to identify individual organisms, were most com-monly seen.

Rehydration method. To test the effect of rehydration ofthe protoplasts by the gradual dilution of the polymer ratherthan the rapid rehydration which occurs when the PEG iseliminated by centrifugation and the cells are resuspended inTCS (26), we incubated the protoplasts with PEG and thenadded aliquots of TCS at 5-min intervals to increase thevolume by four. A control was carried out with one-stepelimination of PEG. TEM studies showed that gradualrehydration enhanced internalization by about 10%, as com-pared with sudden rehydration.PEG solvent. We also studied the effect of the PEG solvent

by using aqueous solutions of PEG instead of TC-PEG. Theonly significant differences that emerged were that in aque-ous PEG solutions, a high percentage of the protoplastsharboring one bacterium lost electron density (Fig. 2b).

Viability of yeast protoplasts after PEG treatment. Proto-plast regeneration was measured both with our experimentalprocedure and with the method of Yamada and Sakaguchi(26). Regeneration of controls was always comparable tothat in a typical fusion process. With our conditions, theregeneration rate was reduced by 60 to 70%, while with theconditions of Yamada and Sakaguchi, it was reduced by only5 to 7%.

Internalization mechanism. To discover the exact nature ofthe internalization mechanism, we carried out TEM analysesof samples during various stages of treatment (incubationwith PEG, rehydration, and washing with TCS). The obser-vations revealed that during the incubation period, largeyeast-bacterium aggregates were formed (Fig. 3a and b) andtwo different situations occurred: (i) bacterial cells stuck tothe plasma membrane of protoplasts when the onset ofinvagination was evident (Fig. 3c) and (ii) several protoplastsmore or less surrounded a bacterial cell (Fig. 3d). Duringrehydration, the size of the aggregates diminished. Thebacteria were mostly contained within invaginations of var-ious sizes in the plasma membrane of the protoplasts (Fig.4a). Some bacteria were lodged deeper inside, with theoutermost tips of the invagination about to join together (Fig.4b). Bacterial cells surrounded by two or three protoplastsinvolved in the process of fusion (Fig. 4c) were also seen, butless frequently. During washing with TCS, there was a sharpdecline in the number and size of the yeast-bacterium

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FIG. 2. (a) S. cerevisiae protoplast harboring three E. coli cells (B). (b) S. cerevisiae protoplast harboring a single bacterial cell (B). Theyeast protoplast has lost electron density, while the E. coli cell remains unaltered. N, Nucleoid; Cw, cell wall; Pm, plasma membrane.

aggregates. At the same time, the protoplasts, whethercontaining a bacterium or not, became heterogeneous insize, probably because of fusing together. At this stage, itwas common to find bacteria lodged in membrane vesicles inthe periphery of the protoplasts (Fig. 4d).

Experiments with A. nidulans. To determine the efficiencyof our method with a different combination as well as todetermine the influence of protoplast size and PEG concen-tration, we repeated the experiments with E. coli-A. nidu-lans, the protoplasts of which are somewhat larger. Weassayed PEG concentrations of 15 to 30%. Although theoptimum PEG concentration for internalization remained15%, at 30% PEG some protoplasts still captured bacteria.The general morphology of the A. nidulans protoplastscontaining bacteria was the same as that of the S. cerevisiaeprotoplasts containing bacteria (Fig. 5). Viability was deter-mined in the same manner as that used for the previousexperiments. Viability was diminished by 1 order of magni-tude, as compared with the control.

DISCUSSION

The primary difficulty involved in determining the effi-ciency of this type of method is to find the most accurate wayof detecting and counting the bacteria inside the protoplasts.We rejected the indirect methods that have been used in

the past, such as tagging a bacterial component (26), becauseit cannot be guaranteed that all of the free bacteria, partic-ularly those entrapped in the aggregations induced by PEG,will be rinsed out. This leaves a choice between examinationwith an electron microscope, a completely reliable methodbut one which does not permit the quantification of theresults, or DAPI staining and examination with an opticalfluorescence microscope (13). The problem with the lattertechnique is that the low resolution afforded by opticalmicroscopy makes it impossible to locate accurately theprokaryotes lodged within the isolated protoplasts and dif-

ficult to identify them among the cell aggregations that buildup as a result of PEG treatment. This means that the onlypossible way of detecting bacteria within fungal protoplastswith any certainty is by TEM, but this method is not veryreliable for arriving at exact quantitative calculations.Our TEM results coincide with those of Yamada and

Sakaguchi (26) only as to the optimum PEG concentration of15% and the yeast/bacterium ratio of 1:100. With our exper-imental conditions, the frequency of internalization in-creased spectacularly. Although the results cannot be math-ematically quantified because of the limitations imposed byTEM, it is possible to state that with the conditions ofYamada and Sakaguchi, it was extremely difficult to locateprotoplasts containing bacterial cells. With our conditions,however, at least one in every five protoplasts played host toa minimum of one prokaryote and, in fact, this percentagemust have been even higher because a bacterium may not bevisible in a protoplast because the plane of cutting may missit.With regard to the changes made and their effect on the

internalization frequency, it is worth pointing out that themost significant increase occurred when PEG was added topellets instead of to suspensions. This result emphasizes thedirect relationship which exists between the degree of aggre-gation and the level of internalization.The existence of an optimum yeast/bacterium ratio (1:

100), below and above which internalization diminishesconsiderably, suggests that there is a relationship betweenthe frequency of internalization and both the degree ofaggregation and the composition of the aggregates. Below aratio of 1:10, for example, the aggregates are composedmainly of protoplasts and there is consequently a tendencyfor these to fuse together. Above a ratio of 1:1,000, however,the tendency is for the bacterial cells to surround theprotoplasts, making it difficult for the latter to fuse togetherand capture bacteria. The pressure exerted by the bacteria in

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NF>V** s~~~~~~~~~..>. ^ ~~~

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FIG. 3. Incubation with PEG. (a and b) General view of the large yeast-bacterium aggregates obtained. The arrows indicate bacterial cellsentrapped between protoplasts. V, Vacuole. (c) E. coli cells (B) pressing around the plasma membrane of a protoplast. (d) Highermagnification of panel b. A bacterial cell is entrapped between yeast protoplasts. The arrows indicate possible points of membrane fusion.

all directions would also make the invagination process inthe protoplasts more difficult.No internalization was detectable when 30% PEG solu-

tions were used with S. cerevisiae and E. coli, a result whichcoincides with the results of Matsui et al. (18), who reportedless internalization in the Vinca rosea-E. coli combinationwhen 20% PEG was used instead of 10% polyvinyl alcohol(PVA). They attributed this phenomenon to the drasticdehydration brought about by the high polymer concentra-

tion, which would result in a reduction of the diameter of thecytoplasmic layer around a large central vacuole, frequentlythinner than the diameter of an E. coli cell.

Nevertheless, in our experiments the typical central vac-uole of the yeast cell was reduced in diameter or evendisappeared during treatment with PEG. For this reason, it isalso likely that extreme dehydration will reduce the elasticityof the cytoplasm and thus make invagination more difficult.We also found that there was a high level of internalization

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FIG. 4. (a, b, and c) Images obtained during rehydration. (a) General view of yeast-bacterium aggregates. B, Bacterial cell; V, vacuole;Nu, nucleus. (b) A single protoplast containing a bacterial cell lodged in a deeper invagination of the plasma membrane; the outermost tipsof the invagination (indicated by arrows) are about to join together and seal the invagination. N, Nucleoid; Cw, cell wall. (c) A bacterial cellentrapped between two protoplasts involved in a fusion process. The arrows indicate the points of the protoplast membranes in close contact.(d) Image obtained during washing with TCS. Yeast protoplasts contained bacterial cells lodged in membranous vesicles. Pm, Plasmamembrane; V, vacuole; Cw, cell wall; N, nucleoid.

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FIG. 5. A. nidulans protoplast harboring bacteria. V, Vacuole;Mi, mitochondrion.

when PEG was eliminated slowly by gradual dilution in thewashing buffer. This result appeared to favor the develop-ment of invaginations during the incubation period. On theother hand, rapid rehydration produced by centrifugationand resuspension of the cells in TC resulted in the rejectionof the bacterial cells from the protoplast invaginations.The polymer solvent does not seem to have any significant

effect on internalization. Nevertheless, the obvious loss inelectron density of host protoplasts, indicating their degen-eration and death, recommends the use of a buffered solutionfor the polymer. The loss of electron density of cells inaqueous PEG may be due to osmotic lysis, as the PEGconcentration used is at the limit for the preservation ofosmotic integrity (22), an effect which is counteracted by theconcentration of Ca2" ions when TC is used. Neither can itbe ruled out that the bacterium itself within the protoplastcontributes to its host's death, especially as the loss ofelectron density occurs in a protoplast which has captured aprokaryote.

This hypothesis is also confirmed by our viability tests, sothat the drastic reduction in the regeneration rate that wefound may be taken as a indication of the efficiency of ourmethod, since the viability of the host decreased as thenumber of internalized bacterial cells increased. Along thesame lines, other authors (19, 26) have suggested that theintracellular presence of the bacterium in the protoplastcauses its disintegration and death, but the final cause of celldeath remains unclear and should be the object of furtherstudy.The TEM images obtained from experiments designed to

find out more about the internalization mechanism show thataggregates of protoplasts and cells form during incubationwith PEG and that internalization takes place mainly duringrehydration; for this reason, it is advisable to rehydrategradually.The internalization of the bacteria into the protoplasts

comes about both by entrapment and by an endocytosis-likemechanism, the latter being the most usual, favored by

aggregate formation which presses the participants togetherand encourages invagination.

Finally, with respect to the experiments carried out withA. nidulans-E. coli, we found that the conditions determinedto be optimum for S. cerevisiae-E. coli are also efficient inthis case. The facts that A. nidulans captures E. coli cells in30% PEG solutions but that S. cerevisiae does not mayindicate a relationship between the larger size of the A.nidulans protoplasts and the polymer concentration which iscapable of inducing internalization. Therefore, the viabilitydata that we obtained with this filamentous fungus confirmthe idea that the presence of the bacterium induces the deathof the protoplast.

ACKNOWLEDGMENTS

We thank our colleague J. Trout for improving the English text.This work was supported by a grant from the Junta de Andalucia.

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