Application of protoplast fusion technique to genetic recombination of Micromonospora rosaria

Application of protoplast fusion technique to genetic recombination of Micromonospora rosaria K. S. K i m a n d D e w e y D. Y. R y u * t The Korea Advanced Institute o f Science, P. 0. B o x 150, Chung-Ryang-Ri, Seoul, Korea

a n d S. Y. Lee Korea University, Seoul, Korea

(Received 6 July 1982; revised 20 December 1982) The optimum conditions for efficient formation and regeneration o f Micromonospora rosaria protoplasts have been determined. The state o f inoculum culture and stage o f growth in a medium containing partially growth-inhibiting concentrations o f glycine had significant effects on protoplasting. A high frequency o f regeneration was accomplished with a hypertonic regeneration agar medium. A slight difference was found in the optimum culture age for formation and regeneration o f protoplasts. Protoplast fusion was carried out using these optimum conditions. The recombinant frequency varied from O. 7 to 5.9% in the intraspecific crosses employing single and multiple auxotrophic markers. Electron microscopy showed stable and intact protoplasts when they were prepared with a hypertonic buffer. However, many protoplasts were shown to be damaged and many membraneous vesicles were observed when prepared in buffer without sucrose. The fusion process o f protoplasts o f Micromonospora was observed with the aid o f electron microscopy. Keywords: Antibiotics; protoplast fusion; genetic recombination; Micromonospora rosaria

Introduction Several methods of genetic recombination have been reported for actinomycetes: transduction, 1 transformation, and conjugation. 2'3 Despite the prevalence of genetic techniques for actinomycetes, few studies reported have been dfiected towards strain development, due possibly to the laborious and time-consuming introduction of genetic markers needed for the selection of recombinants, and the associated deleterious side effects. Therefore, industry has primarily employed conventional random mutagenesis for strain improvement. More than 3000 antibiotics have been described, of which 2080 are actinomycete antibiotics. 4 New antitumour agents, as well as antibacterial agents, are still in great demand. The increasing drug resistance of microbial strains has led to a greater need for novel antibiotics. From this point of view, protoplast fusion is considered to be a most powerful and promising technique for the genetic manipulation of industrial microorganisms, because it induces not only a high frequency of recombination but also genetic interactions between different microorganisms: interspecific,s'6 intergeneric, 7 and even between different kingdoms, s Although the formation of protoplasts had already been reported in the 1950s, 9,1° protoplast fusion by poly*To whom correspondence should be addressed. tPresent address: University of California, Davis, CA 95616, USA. 0141--0229/83/040273--08 $03.00 © 1983 Butterworth & Co. (Publishers) Ltd

(ethylene glycol) (PEG) was not an established technique until the 1970s. This technique has now been successfully applied to fungi, 11 yeast 12 and bacteria, 13 and more recently it has been extensively used for the genetic investigation of the genus Streptomyces. 14-19 To expand this technique as a general method in actinomycetes, we initiated studies cn genetic recombination using Micromonospora rosaria, a producer of rosaramicin.2° Beretta et al. demonstrated genetic interactions between two auxotrophs ofMicromonospora spp. which resulted from an intraspecific recombination. 2 Protoplast fusion o f Micromonospora was first successfully carried out by Szvoboda e t al. 21 But genetic studies o f Micromonospora, a genus of major commercial importance which produces a variety of novel antibiotics, have been largely ignored. The paucity of genetic studies on this organism may be at least partially due to their physiological sensitivity to environmental factors and their limited sporulation. It is considered to be of some importance to apply the protoplast fusion technique to organisms belonging to this group to further the understanding of the genetic properties of this genus and, ultimately, to obtain superior antibiotic-producing strains. This is the first original report on the protoplast fusion and genetic recombination of rosamicin-producing M. rosaria strains. Conditions for efficient protoplast formation and their regeneration into mycelial forms were established to

Enzyme Microb. Technol., 1983, Vol. 5, July

273

Papers obtain a high frequency of genetic recombination. Morphological studies on protoplasts, mycelia and fusing protoplasts ofMicromonospora rosaria were carried out using transmission electron microscopy. Materials and m e t h o d s

Organism and rnutagenesis M. rosaria NRRL 3718 was maintained in lyophilized vials. Auxotrophic derivatives were induced by an N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis using mycelial fragments as well as protoplasts. M. rosaria cells were harvested by centrifugation at an early stationary phase (~5.5 g cell/litre) of a culture grown in GER medium (see Media, below), sonicated (7 kilocycles, 100 watts, 1 rain, Ultrasonic Dismembrator, Model 300), washed with 0.1 M Tris-maleic acid (TM) buffer (pH 7.2) and centrifuged (3000g for 10 rain). After suspending the pellets in TM buffer, freshly prepared MNNG solution (1 mgml -~) was added to the suspension to give a final concentration of 0.3 mg m1-1. This suspension was incubated for 10-20 min at 30°C, centrifuged, and resuspended in 0.1 M sodium phosphate buffer (pH 7.2). These mutagenized mycelial fragments were used as the new inoculum and cultured for 2 days at 30°C in GER medium. This culture was again sonicated and plated on M40 and GER agar media after appropriate dilutions for plating. Ampicillin treatment (at 30 gg ml -a after allowing a 6 h lag period for phenotypic expression) was employed for enrichment of auxotrophs. Mutant strains used in this study are listed in Table 1. No significant change in the growth characteristics of these auxotrophic mutants was observed. Protoplasting ofM. rosaria MR 217 was somewhat retarded and a longer lysozyme (mucopeptide N-acet ylmuramoylhydrolase, EC 3.2.1.17) treatment (4 h) was required. This strain showed red-brown pigmentation. Reagents PEG (MW 1000, 4000 and 6000), glycine, lysozyme and lytic enzyme were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tris(hydroxymethyl)aminomethane (Tris) was from Merck and Co. (Rahway, NJ, USA) and N-Tris(hydroxymet hy0met hyl-2-aminoet hanesulphonic acid (TES) from Pierce Chemical Co. (Rockford, IL, USA). All other chemicals used were reagent grade. Table 1 Mutants of M, rosaria N R R L 3718 employed

Strain

Genetic marker

M. rosaria

MR MR MR MR MR MR MR MR MR MR MR

4 8 18 20 30 28 23 210 212 217 221

Prototroph ade arg trp lys asp his trp ade trp ade lie a d e h i s trp argura arg his

Antibiotic production a + + + + + + -+ + + + +

Source N R R L 3718 NRRI_ 3718 N R R L 3718 N R R L 3718 N R R L 3718 N R R L 3718 MR 18 MR 4 MR 4 MR 4 MR 8 MR 8

a +, Rosamicin biosynthesis is fully expressed in the range 25--30/zg m1-1 broth in our culture conditions; --, No production of rosamicin; -+, Rosamicin biosynthesis is partially expressed in the range 5--10p, g ml -~

274

Enzyme Microb. Technol., 1983, Vol. 5, July

Media A complex medium GER 2° contained (g l-L): beef extract, 3; tryptone, 5 ; dextrose, 1 ; soluble starch, 24; yeast extract, 5; CaCO3, 2; pH adjusted to 7.6. Synthetic agar medium (M40 agar medium), 22 contained the following, in g per litre distilled water: KH2PO4, 1 ; MgSO4 • 7H20, 0.05; (NH4)2SO4,3.5 ; L-asparagine, 1.5 ; D-glucose, 10; 1 ml each of solutions A, B and C (see below); agar, 20. Solution A contained 10 g NaC1 in 100 ml distilled water; solution B, 10 g CaC12 • 6H20 in 100 ml distilled water; solution C, 50 mg H3BO3,4 mg CuSO4" 5H20, 10 mg KI, 20 mg FeC13 • 6HzO, 40 mg MnSO4" H20, 40 mg FeSO4" 7H20, 20 mg (NH4)6Mo7024" 4H20, in 100 ml distilled water. Cell concentration was determined from dry cell weight measurements. Cells were collected and washed twice with TM buffer by centrifugation, then dried overnight in an oven at 105°C, and weighed. Media for protoplast formation and regeneration used were similar to the Medium P and Medium R1, respectively, used by Okanishi et al.23 The regeneration agar medium (RM) that we used for M. rosaria has the following composition: sucrose, 125 g; K2SO 4, 0.25 g; trace element solution, 1 ml; MgC12 • 6H20, 5.09 g; D-glucose, 10 g; L-asparagine, 4g; casamino acid, 0.1 g; agar, 20 g, d-H20 to make 700 ml; CaC12 • 2H20, 7.37 g; d-H20 to make 100 ml; KH2PO4, 0.05 g, d-H20 to make 100 ml; 0.25 M TES buffer, pH 7.6, 100 ml. The composition of the trace element solution was the same as that defined by Okanishi et aL23 The solutions were sterilized separately and then combined. The only changes in the modified medium P that we used were (g 1-'): MgC12" 6HzO, 5.09; CaC12 • 2H20, 7.37; the pH was adjusted to 7.6. Preparation and examination o f protoplasts Mycelia ofM. rosaria at an early stationary growth phase grown in GER medium were sonicated and used to inoculate (5% v/v) GER medium containing glycine. Harvested cells (10 ml) were washed with Medium P, treated with lysozyme (2 mg m1-1 ) and incubated at 32°C. If mycelial fragments were not visible under a microscope, the suspensions were mixed gently by a pipetting action to complete protoplasting. Counting of protoplasts was achieved by two methods: (1) direct counting using a haemocytometer under a phasecontrast microscope; (2) counting after staining of protoplasts. In this method, Gram's crystal violet solution containing 0.3 M sucrose was used to stain the appropriately diluted suspension of protoplasts at a concentration of 0.015 ml m1-1 after filtering through a Millipore filter (0.45 ~m). The stained protoplasts were counted with a haemocytometer under a light microscope. Regeneration o f protoplasts The regeneration agar medium defined by Okanishi et al.23 was used as the basic medium forM. rosaria. The prepared suspension of protoplasts was diluted with medium P and spread on agar media and incubated at 32°C. The plates were observed at two-day intervals until no new colonies appeared. Colonies from non-protoplasted cells were examined by diluting the protoplasts in distilled water and plating on regeneration agar medium. Standard crossing experiment A conventional crossing method 24-26 was employed using mycelial fragments because of the non-conidia forming

Protoplast fusion o f

properties ofM. rosaria. Concentrated cell suspensions of each auxotrophic strain were prepared by ultrasonication, centrifugation and resuspension in TM buffer. A 0.1 ml aliquot of this suspension (containing ~1 x 108 CFU of each strain) was inoculated onto a GER agar slant and incubated at 32°C for 7 to 10 days. Cells were scraped and suspended in 10 ml TM buffer. These suspensions were sonicated and filtered. Then, 0.1 ml aliquots of suitable dilutions were spread on M40 medium with or without the corresponding nutritional requirements. After incubation for 5 to 7 days at 30°C, the frequency of recombinants was determined.

S. Kim et aL

9

9= -

4

oa-

c) 0

Genetic fusion by PEG Equal volumes (0.5-1.0 ml containing ~ 2 x 10 9 protoplasts) of protoplast suspensions of two auxotrophic strains were mixed, and after centrifugation (5000 rev/min for 10 rain, equivalent to 3000 g, Sorvall RC-5B) the supernatant was decanted. The pellets were resuspended in 0.1 ml medium P and gently mixed with 0.9 ml PEG 1000 solution (55.6%, w/v) to make 50% PEG (w/v). Control pellets were resuspended in 1 m] medium P. After incubation at 32°C for 3 rain with intermittent gentle mixing by hand, the suspensions were diluted in medium P, and 0.1 ml aliquots of appropriate dilutions were plated on regeneration agar media with or without the corresponding nutritional requirements. Incubation was carried out at 32°C for 10 to 12 days.

Micromonospora rosaria: K.

2

4

I

6

8

Culture Age (Days) Figure 1 Effect of growth phase and culture age on protoplast formation. Each sample contained the same amount o f cells. Curves show the counts of protoplasts when lysozyme was treated for: ®, 1.5 h; o, 5 h. cz, Dry cell weight

6

2.0

'~ •i--- 0

Genetic analysis o f recombinant colonies In order to confirm that the prototrophic colonies were true recombinants, colonies were picked, inoculated onto slants of GER agar medium and incubated at 32°C for 4 days. The grown cells were scraped from the slants, washed twice in TM buffer and centrifuged. After sonication of mycelia, the suspension was filtered and 0.1 ml aliquots of appropriately diluted suspensions were plated on M40 agar medium supplemented with all growth factors. After incubation for 5 days, colonies were randomly picked and plated on M40 agar media supplemented with or without various combinations of growth factors. Colony formation and growth on these plates was evaluated to identify prototrophic colonies.

Preparation o f specimens for electron microscopy Specimens were fixed with 3% (v/v) glutaraldehyde. Throughout our experiments, 0.1 M sodium phosphate buffer (pH 7.4) containing 10% (w/v) sucrose was used. The mixture was gently stirred, allowed to stand overnight in a refrigerator, washed twice with the buffer and centrifuged. These pellets were suspended in warm liquid agar (2% w/v, in buffer) and solidified. Agar blocs were then cut on a glass slide into pieces ~ 1 mm square. These blocks were suspended in osmium tetroxide (1%), allowed to stand for 2 h and rinsed with the buffer solution. Dehydration was achieved with a series of alcohols of increasing concentration and the blocks were then embedded in Epon-812. Sections were cut on a Sorvall MT-2 ultramicrotome with glass knives. The sections were stained with uranyl acetate and lead acetate, and examined with an electron microscope (Hitachi H-500, Japan). For comparison, a phosphate buffer containing no sucrose was used.

Results

4.0

C3

0%

n: T~ O

i 0.025

i O.O5

i 0.075

,T O.t

Glycine Concentration (%, w/v) Figure2 Effect of glycine concentration on growth and protoplast formation. Cells were harvested after 48 h cultivation and treated w i t h lysozyme for 2 h. e, Dry cell weight; o, counts of protoplasts

or in a defined medium, although chlamydospores are often found in the middle of mycelia. For the efficient preparation of protoplasts, the growth phase was investigated first. Figure 1 shows that protoplast formation was affected by the culture age. Cells from an early stationary phase were the most susceptible to lysozyme treatment. Cells from the late stationary phase were less readily converted to protoplast, even after prolonged treatment with ]ysozyme, e.g. > 2 4 h. For the preparation of protoplasts ofM. rosaria, glycine was found to be most effective at a concentration of 0.075% (w/v) (Figure2). However, cell growth was completely inhibited at 0.15% (w/v) glycine concentration. The state of the inoculum was also very important for efficient protoplasting. Ultrasonication of the inoculum for >~1 rain with the ultrasonic dismembrator, as described above, led to a two-fold increase in protoplasting efficiency (Figure 3). Mycelial growth was slightly inhibited by ultrasonication of the inoculum. When the appropriate combinations of these methods were employed for protoplasting, mycelial fragments were rarely found after 50 rain of lysozyme treatment without the addition of another lytic enzyme.

Conditions for protoplast formation M. rosariagrows as a long and fine filament with multiple

Regeneration o f protoplasts into mycelial form As shown in Table 2, for regeneration a slightly alkaline

branches. It does not produce conidia in a complex medium

range of pH with TES buffer was the most effective. The

Enzyme Microb. Technol., 1983, Vol. 5, July

275

Papers

reversion excludes the possibility that the prototrophs resulted from spontaneous reversion. A high frequency of prototrophs by protoplast fusion in 4 to 5-factor crosses of auxotrophic mutant strains was achieved (Table 5). On the other hand, conventional crossing gave a lower, by three to four orders of magnitude recombinant frequency. Tables 4 and 5 also show the results of control experiments. In each cross, the spontaneous reversion frequencies of protoplasts during regeneration are insignificant compared with those from recombination by protoplast fusion. When prototrophic colonies, which appeared on regeneration media after protoplast fusion, were analysed no auxotrophic segregant was detected among more than 300 colonies which originated from 10 randomly picked prototrophic colonies. This result suggests that the prototrophs obtained here are recombinants.

~0

0

o."-

1

T o

I

I

I

t 2 3 Sonication Time(rain.)

Figure 3 Effect of sonication of inoculum culture on protoplasting. Cells were grown in GER medium containing 0.075% (w/v) glycine and harvested after 48 h cultivation. Lysozyme treatment was for 1 h at the concentration of 2 mg m1-1. Each sample contained the same amount of cells

Studies using electron microscopy Thin sections of intact mycelia and protoplasts of

M. rosariawere prepared and their microstructure was Table 2 Effect of buffers and pH on regeneration efficiency A

TES buffer 6.8 7.2 7.7 8.3 Tris buffer 6.8 7.2 7.7 8.0 8.7

0.17 11.8 22.1 8.9

¢-

¢C3n 30

20

2 0.13 6.8 18.4 17.1 9.7

regenerated colonies usually appeared after 7 to 8 days and no more colonies appeared after 12 days. The optimum temperature for regeneration was found to be 32°C. The growth phase of cells before harvesting had a significant effect on the regeneration efficiency (Figure 4) as well as on the protoplasting efficiency (Figure 1). The regeneration was most efficient when cells from the early stationary phase were used. The best culture age for regeneration is slightly different from that for protoplasting. The optimum concentrations of KH2P04 and sucrose for regeneration were 0.025% (w/v) and 12.5% (w/v), respectively. Of the amino acids tested, 0.4% L-asparagine gave the highest regeneration frequency. The combination of Mg2÷ and Ca 2+ ions that supported the highest frequency (30%) of regeneration was 50 mM MgC12 and 25 mM CaCI2 (Table 3).

Genetic recombination by protoplast fusion The frequency of spontaneous reversion to prototrophy of auxotrophic strains ofM. rosariawas ~10 -6 per CFU or less. The real frequency may be much less than this value because plate counting was performed with mycelial fragments. When protoplast treatment with PEG 1000 was performed, fusion occurred at a frequency of about 1-6% (Table 4). A low frequency (I0-6/CFU) of spontaneous

E n z y m e M i c r o b . T e c h n o l . , 1983, V o l . 5, J u l y

O" LL

0

Cells were harvested after 2.5 days incubation a Each buffer, 0,25 M, was made and cold-sterilized by Millipore filtration (0.45#m), and then combined with the other components as described by Okanishi et al. 23 b Regeneration efficiencies were defined as the fraction of protoplasts that are regenerated into colonies on the regeneration agar media

276

6

Regeneration efficiency after 10 days (%)b

PHa

r~

tO

0

I

2

3

Culture

4

5

6

co o ~c-

w

Age (Days)

Figure 4 Effect of growth phase on the efficiencies of regeneration. o, Dry cell weight; a, regeneration efficiencies Table 3 Effect of concentration of Mg 2+ and Ca 2+ on regeneration efficiency

Regeneration efficiency (%) Mg (mM)

Ca (mM)

After 7 days

After 10 days

0 5 25 10 25 50 50

0 5 10 25 50 25 50

18 21 20 22 23 25 17

20 21 21 23 29 30 19

Table 4 Frequencies of prototrophs by protoplast fusion

Cross (genotypes)

Control (without PEG)

Frequencies of prototrophs with PEG 1000 a (50%, w/v)

MR 18 + MR 20 (trp) (lys) MR 18 + MR 4 (trp ) (ade ) MR 18 + MR 30 (trp ) (asp)

1.5 X 10 -5

3.2 X 10 -2

3.7 X 10 -s

1.3 × 10 -2

9.03 X 10 - 6

5.9 X 10 -5

a This frequency is the ratio of the number of prototrophic colonies grown on regeneration agar medium to the total number of colonies grown on supplemented regeneration agar medium

Protoplast fusion o f Micromonospora rosaria: K. S. K i m et aL Table 5 Fusion frequencies between multiple marker auxotrophs by protoplast fusion and standard crossing

(control)

Recombinant frequencies by standard crossing (per CFU)

4.3 X 10 -2

7.0X 10 -7

1.4X 10 -4

1.1 X 10 -2

2.3 X 10 -6

N.t.

2.1X 10 -2

1.4X 10 -6

2.4X 10 -6

0.7 X 10 -2

3 . 1 X 10 -6

2.8X 10 -6

Cross (genetic markers)

Prototroph frequencies with PEG treatment

MR217

(arg ura) MR 221

+MR28

(his trp) + MR 23

(arg his) MR212

Prototroph frequencies without PEG treatment

(trp ade) + MR217

(trp his ade) (argura) MR210

+ MR 221

(lie ade)

(are his)

N.t., not tested

studied under an electron microscope. Figure 5 shows that the cell wall of mycelia was ~28 nm thick and mesosomes of lamella-like structure were found to be connected to the cell membranes. Nuclear regions cannot be easily distinguished in mycelial sections. In the protoplasts, neither cell walls nor mesosomes of lamella-like structure were observed (Figure 6). Most of the protoplasts were stable in their structure, and their cell membrane, cytoplasm, and nuclear regions could be identified (Figure 6). In some protoplasts the nuclear regions were localized and condensed, but in others they were rather dispersed and showed a large variation in size. This is probably not due to a swelling phenomenon since the protoplasts prepared in medium P, which contained from 1 to 20% sucrose, were

unchanged. In addition, ghosts and demembraned protoplasts were also observed. When a buffer not supplemented with sucrose was used for the preparation of ultrathin sections of protoplasts, they showed unstable structures (Figure 7). Threads of DNA were observed between protoplasts, and membraneous vesicles were abundant, but were rarely found in sections prepared with hypertonic buffers (Figure 6). Their prevalence in unstable protoplasts indicates that they are probably reassembled structures of membranes. Partially broken membranes were often found in these sections (Figure 7). When sections of protoplasts treated with PEG 1000 were prepared, the ultrastructure of fusing protoplasts could be observed (Figure 8). Fusion of two nuclear regions was noticeable. No fusion bodies were observed in the control experiment without PEG.

Discussion

E

=L

Figure 5 Electron micrograph of sections of intact mycelia of M. rosaria: W, cell wall; M, mesosome

Using M.. rosaria, the important factors that affect protoplast formation were studied first. As demonstrated by other authors who used Streptomyces spp., we found M. rosaria to be highly sensitive to lysozyme when grown in a medium containing glycine. But its sensitivity to glycine was found to be about ten times higher than for most Streptomyces spp. reported previously, 16 and cell growth was completely inhibited in the presence of 0.15% (w/v) glycine. Szvoboda et al. showed that Micromonospora echinospora and M. inoyensis were more sensitive to glycine treatment than Streptomyces. 21M. rosaria also showed higher sensitivity against MNNG treatment in our mutagenesis experiments. The best protoplasting was achieved when the cells were harvested in an early stationary phase of a culture. Under optimal conditions, the mycelia ofM. rosaria were almost completely converted to stable protoplasts within 1 h with lysozyme treatment. As found for other streptomycetes, 14,24 high efficiencies of protoplasting and regeneration are essential to the genetic manipulation ofM. rosarict In our preliminary experiments, the R1 medium used by Okanishi et aL was found to be best suited for the regeneration of protoplasts ofM. rosaria as compared to the regeneration agar medium used for other actinomycetes.23,27 The regeneration ofM. rosaria protoplasts was highly susceptible to environmental conditions, and great care was needed for its regeneration. It was also found that there is a slight difference in the optimum culture age for protoplasting and regeneration (Figures 1 and 4). The composition of the regeneration agar medium used by Okanishi et al. 23 had to be slightly modified for M. rosaria. Slight alkalinity (pH 7.6-7.7) of the regeneration medium seems to favour good regeneration of Micromonospora protoplasts, based on the findings of Szvoboda et al.21 and

E n z y m e M i c r o b . T e c h n o l , , 1983, V o l . 5, J u l y

277

Papers

Figure 6 Transmission electron micrograph of protoplasts prepared with hypertonic buffer. Intact protoplasts were observed with dispersed and localized nuclear region. They show large size variation. CM, Cytoplasmic membrane; N, nuclear region; G, ghost; MP, miniprotoplast

Figure 7 Transmission electron microscopy of protoplasts prepared with buffer without sucrose supplement. Many protoplasts were damaged and membraneous vesicles were frequently observed. CM, Cytoplasmic membrane; N, nuclear region; MV membraneous vesicles; T, threads of DNA

278

E n z y m e Microb. Technol., 1983, Vol. 5, July

Protoplast fusion o f M i c r o m o n o s p o r a rosaria: K. S. Kim et aL

Figure 8 Electron micrographs of protoplasts treated with PEG for 5 rain. Protoplasts were derived from two strains (MR217 & MR28) of

M. rosaria before PEG 1000 treatment

ourselves (Table 2). The regeneration process ofM. rosaria protoplasts was relatively slow (7 to 12 days) and asynchronous, and growth inhibition by early colonies was not significant. About 30% of the protoplasts were regenerated into mycelial forms on RM medium and, among these colonies, less than 0.5% were from non-protoplasted cells. Ultrathin sections of intact mycelia ofM. rosaria had the structure typical of Gram-positive cell wall. Mesosomes were frequently observed and shown to be connected with the cytoplasmic membrane (Figure 5). When prepared with hypertonic buffer, most protoplasts remained intact. It was noted that there was a large variation in size, and small protoplasts with diameter <0.4/am had no nuclear region. Mesosome of lamellalike structure could not be detected in protoplasts (Figure 6). Their electron micrographs were very similar to those o f S t e p t o m y c e s griseus, 23 A very brief exposure to the phosphate buffer without sucrose during specimen preparation led to extensive damage of the protoplasts (Figure 7). Vesicles with membrane-like layer were frequently observed in these samples. These vesicles may come from the damaged cytoplasmic membrane, but might also have originated from the released mesosomes. 23 Some structural correlations between the membraneous vesicles and the cytoplasmic membrane were often observed. Okanishi et aL 23 indicated that the concentration o f Mg 2+ and Ca 2+ has some effect on the formation of vesicles. Resealing and repairing o f membraneous structures have also been reported elsewhere. 28 The electron micrographs of protoplasts treated with PEG (Figure 8) frequently showed interactions between protoplasts. Protoplasts can be induced to fuse by treatment with PEG 1000. When protoplasts of a mutant strain with single and multiple auxotrophic markers were fused the

recombinant frequency achieved was about 1 - 6 % (Tables 4 and 5). This high frequency of prototrophic recombinants in crossing of various auxotrophic pairs indicates that recombination frequently occurs at multiple sites when M. rosaria protoplasts are fused. Recently, protoplast fusion techniques have been applied to the elucidation of gene organization 17 as well as in the strain development of antibiotic producers. 5 The technique of protoplast fusion forMicromonospora is being optimized to obtain hybrid strains for overproduction or the production o f novel antibiotics and possibly to achieve interspecific and/ or intergeneric recombination. The results show that there is great promise for the protoptast fusion technique as an essential tool for genetic studies of non-conidia forming actinomycetes such as M. rosaria. References 1 Stuffard, C.J. Gen. Mierobiol. 1979, 110, 479-482 2 Beretta, M., Betti, M. and Polsinelli, M. J. Bacteriol. 1971, 107,415-419 3 Hopwood, D. A. and Merrick, M. J. Bacteriol. Rev. 1977, 41,595-635 4 B~rdy,J. Adv. Appl. Microbiol. 1974, 18, 309-406 5 Fleck, W. F. in Genetics oflndustrialMicroorganisms (Sebek, O. K. and Laskin, A. I., eds) American Society for Microbiology, Washington, DC, 1979, pp. 117-122 6 Godfrey, O., Ford, L. and Huber, M. L. B. Can. J. Microbiol. 1978, 24,994-997 7 Wesseling,A. C. and Lago, B. D. Dev. Ind. MicrobioL 1980, 22, 641-651 8 Ward,M., Davey, M. R., Mathias, R. J., Cocking, E. C., Clothier, R. H., Balls, M. and Lucy, J. A. Somatic Cell Genet. 1979, 5,529-536 9 Bradly, S. G.J. Bacteriol. 1959, 77, 115-116 10 Sohler, A., Romano, A. H. and Nickerson, W. J. J. Bacteriol. 1958, 75,283-290

E n z y m e Microb. Technol., 1983, Vol. 5, J u l y

279

Papers 11 12 13 14 15 16 17 18 19 20

Peberdy, J. F. and Kevei, F. in Protoplasts - Applications in Microbial Genetics (Peberdy, J. F., ed.) University of Nottingham, England 1979, pp. 2 3 - 2 6 Van Soligen, P. and Van der Plaat, J. B.J. Bacteriol. 1977, 130, 9 4 6 - 9 4 7 Foder, K., and Alf'oldi, L. Proc. NatlAcad. Sc~ USA 1976, 73, 2147-2150 Baltz, R. H.J. Gen. Microbiol. 1978, 107, 9 3 - 1 0 2 Baltz, R. H. Dev. lnd. Microbiol. 1980, 21, 4 3 - 5 4 Hopwood, D. A., Wright, H. M., Bibb, M. J. and Cohen, S. N. Nature 1977, 268, 171-174 Hopwood, D. A. and Wright, H. M. Mol. Gen. Genet. 1978, 162, 307-317 Hopwood, D. A. and Wright, H. M. J. Gen. MicrobioL 1979, 111,137-143 Hopwood, D. A. Sci. Am. 1981, 245, 6 6 - 9 3 Wagman, G. H., Waitz, J. A., Marquez, J., Murawski, A., Oden, E. M., Testa, R. T. and Weinstein, M. J. J. Antibiot. 1972, 2 5 , 6 4 1 - 6 4 6

280

Enzyme Microb. Technol., 1983, Vol. 5, July

21

22 23 24 25 26 27 28

Szvoboda, Gy., Lang, T., Gado, I., Ambrus, G., Kari, C., Fodor, K. and Alf61di, I. in Advances in Protoplast Research (Ferenczy, L. and Farkas, G. L. eds), Hungarian Academy of Sciences, Budapest 1980, pp. 2 3 5 - 2 4 0 Polsinelli, M. and Beretta, M. J. Bacteriol. 1966, 91, 63-68 Okanishi, M., Suzuki, K. and Umezawa, H.J. Gen. Microbiol. 1974, 80, 3 8 9 - 4 0 0 Ochi, K., Hitchkock, M. J. M. and Katz, E.J. Bacteriol. 1979, 139, 9 8 4 - 9 9 2 Oh, Y. K., Speth, J. L. and Nash, C. H. Dev. lnd. Microbiol. 1980, 2 1 , 2 1 9 - 2 2 6 Hopwood, D. A. BacterioL Rev. 1967, 3 1 , 3 7 3 - 4 0 3 Sermonti, G. in Genetics o f Antibiotic Producing Microorganisms Wiley Interscience, New York, 1969, pp. 263-315 Racker, E. in Essays in Biochemistry (Campbell, P. N. and Dickens, F., eds) 1970, pp. 1 - 2 2