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Intergenus protoplast fusion between Streptomyces and Micromonospora with reference to the distribution of parental characteristics in the fusants
JOURNALOF BIOKIENCE AND BIOENGINEERING Vol. 88, No. 2, 143-147. 1999
Intergenus Protoplast Fusion between Streptomyces and Micromonospora with Reference to the Distribution of Parental Characteristics in the Fusants CHIAKI IMADA,* MASANORI OKANISHI, AND YOSHIRO OKAMI Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan Received 23 March 1999/Accepted 25 May 1999
The protoplasts of two strains of Mcromonospora which were sensitive to kanamycin (KMS) and utilized raffinose (Raf+), and one strain of Streptomyces griseus which was resistant to KM (KM’) and did not utilize raffinose (Raf-), were prepared, mixed in the presence of polyethylene glycol (PEG) and incubated on regeneration agar plates. Recombinant colonies showing KM’.Raf+ were obtained at a frequency of 2 X 10T6. Their recombinants displayed a signiticant exchange of taxonomic characteristics between the two genera, although the majority appeared similar to the parent Micromonospora in their morphology as well as growth at 4O‘C. Their patterns of utilization of carbohydrates, amino acids and diammonium hydrogenphosphate were different from those of the Micromonospora. Intermediate or novel types which differed from their parents in their tolerance to NaCl and sensitivity to aminoglycoside antibiotics were also observed. Out of the 31 fusants obtained, two showed antimicrobial activity against Bacillus subtilis PC1 219, without any activity against Escherictia coli K-12 or Candida albicans 3147. The active substancemay be a newly formed antibiotic, different from streptomycin in S. griseus. [Key words: protoplast
fusion, Streptomyces,Micromonospora]
aminoethanesulphonic acid (TES) were obtained from Nakalai Tesque Inc. (Kyoto). Lysozyme from chicken egg white and achromopeptidase purified from Achromobatter lyticus were purchased from Wako Pure Chemical Co. (Osaka). All the other chemicals used in the present study were reagent grade. Parental strains M. chalcea IF0 13503 and M. carbonaceae sub sp. aurantiaca KCC-0168 were obtained from The Institute for Fermentation (Osaka), and Kaken Chemical Co. Ltd. (Tokyo), respectively. S. griseus 2C-5 was kindly given by Dr. K. Hotta of National Institute of Infectious Diseases and was maintained in our laboratory. The two former strains were sensitive to KM (2 pg/ml, KM”) and utilized raffinose (Raf+) for growth. S. griseus was resistant to KM (200 fig/ml, KM’) and did not utilize raffinose (Raff). Protoplast fusion The strains employed were cultivated in 100 ml of 172F medium (13) containing 0.2% glycine in a 500 ml Erlenmeyer flask at 27°C on a rotary shaker (200 rpm). The mycelia of these strains at the late logarithmic growth phase (4-6 d) were induced to form protoplasts according to the method of Okanishi et al. (9) with minor modifications. The mycelia obtained from about 10 ml of S. griseus culture were incubated with 0.6 ml of lysozyme solution (40 mg/ml in 0.5 M sucrose), 0.6 ml of achromopeptidase solution (0.2mg/ml in 0.5 M sucrose) and 5 ml of reaction mixture (P3) at 37°C for 3 h, and about 2 X lo9 protoplasts/reaction mixture were obtained. The two strains of Micromonospora were incubated with the same reaction mixture as mentioned above for l-2 d to obtain about 6 x lo9 protoplasts/reaction mixture. The number of protoplasts was counted directly using a Petroff-Hauser bacterial counting chamber under a phase-contrast microscope. Fusion treatment The mixture of protoplasts (approximately 5 X lo* for each) from the two parental strains was centrifuged, and 2ml of PWP medium (14)
Okanishi et al. (1) specified on the culture media, the special conditions required for the preparation of stable protoplasts in actinomycetes, as also on the procedures for regeneration, promoting more protoplast fusion experiments on bacteria (2, 3), actinomycetes (4), yeasts (5), and fungi (6). This has contributed not only to improving the industrially important microbes, but also to a better understanding of the secondary metabolism in antibiotic synthesis. The fusion technique is considered to be a powerful and promising technique for the genetic manipulation of microbes, to induce a high frequency of recombination between different, not only intraspeties but also interspecies microbes (7-lo), and more so between microbes of different kingdoms (11). Subsequently, a new antibiotic obtained by an interspecies cross among Streptomyces was reported by our group (12). The fusion techniques used were greatly improved by Okanishi et al. (9). The recombinants obtained had stable characteristics, and novel antibiotics are expected to be found in recombinants obtained by interspecies protoplast fusion (10). However, no report on the intergenus fusion between different genera of actinomycetes is yet available. This is the first report on protoplast fusion and genetic recombination between one strain of Streptomyces griseus and two strains of different species of Micromonospora. The characteristics of the fusants are described and the distribution of the parental characteristics in the fusants is discussed. MATERIALS
AND METHODS
Chemicals Polyethylene glycol (PEG, molecular weight 1000) and N-Tris (hydroxy methyl) methyl-2* Corresponding author. Present address: Tokyo Univ. Fisheries, 5-7 Konan, Minato-ku, Tokyo 108-8477, Japan. 143
144
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ET AL.
J. BIOXI. BIOENL,
containing 40% PEG 1000 was added to the precipitate, followed by incubation for 3 min at room temperature. The entire contents of the tube were sucked in and out by a Pasteur pipette. The PEG-treated protoplasts were then diluted by the addition of 3 ml of PWP medium. Regeneration after fusion treatment Protoplast regeneration was carried out according to the method of Shirahama et al. (14) using a modified regeneration medium (15). The protoplast suspension obtained by the fusion treatment was serially diluted, plated on MR 0.3s (15) medium for regeneration, and incubated for 14 d at 27-37°C. The colonies developed on the plates were replica-plated onto the selection medium containing 20 pg/ml of KM and 0.5% raffinose in modified SAP-II (6) agar, and incubated for 14 d. SAP-II agar consisted of 900 ml of inorganic solution (0.5 g MgS04.7Hz0, 0.5 ml 10% CaC12.2Hz0 solution, l.Oml TM solution, 1.6 g NH4N03 and 0.5 g K2HP04), 100 ml of 250 mM TES buffer (pH 7.2), and 16g of Agar Noble (Difco). The KM’.Raf+ colonies which appeared were picked and purified three times to remove pseudorecombinants (heteroclones or heterokaryons). They were preserved in SAP-II agar slants containing 20 pg/ml of KM and 0.5% raffinose. Characteristics of the fusant strains The NaCl tolerance of the fusants was tested by the use of SAP-II agar containing 1.0% glucose and varying concentrations of NaCl. Their utilization patterns of carbohydrates and amino acids were determined as follows. Each carbohydrate or amino acid was added to the SAP-II agar in place of glucose or NH4N03 to obtain a final concentration of 1% and 50mM, respectively. The utilization of diammonium hydrogenphosphate (25 mM) in SAP-II agar was also tested in place of NHdN03. Antibiotic resistance was also determined in SAP-II agar containing 1.0% glucose and varying concentrations of the antibiotics employed. Antimicrobial activity The clones were cultured in disposable 24-well plastic plates (Sumitomo Bakelite Co. TABLE
1. a. Distribution
Morphology
Ltd.) on a Minishaker (B. E. Marubishi Co. Ltd., 200 rpm) for 7 d at 27°C according to the method reported previously (16). The medium consisted of 3.Og NaCl, 0.5 g MgS04.7Hz0, 1 ml 10% CaCl* .2Hz0, 1.5 ml TM solution, 0.5 g K2HP04, 15.0 g maltose and 8.0 g Bactosoytone (Difco) in 1 I of deionized water (pH 7.0). Their antimicrobial activities were determined on agar plates seeded with Bacillus subtilis PC1 219, Escherichia coti K- 12 or Candida albicans 3 147. RESULTS Generation of recombinants by protoplast fusion The intergenus fusion treatment, regeneration and replica-plating procedures (described in Materials and Methods section) yielded KM’. Raf+ colonies at a frequency of about 2 x 10 -‘j. The selection medium was SAP-II agar medium containing KM (20 pg/ml) and raffinose (0.5%). In the case of the self-fusion experiments of Micromonospora, KMT. Raf’ colonies were detected as small colonies at almost the same frequency as that following intergenus fusion. However, none of them grew on the selection plate (SKM, +Raf) by subcultivations, indicating that the selected colonies were not true recombinants. In the case of the self-fusion of S. griseus, no KM’.Raf+ colonies were detected. On the other hand, the colonies of KM’.Raf+ obtained by intergenus fusion, did not show any change in the selected characteristics after purification Distribution of parental characteristics in the fusants Morphology, growth at 4O”C, tolerance to NaCl and diammonium hydrogenphosphate utilization In all the experiments of intergenus fusion in this study, almost all fusants were similar to Micromonospora in their morphological appearance as well as their growth ability at 40°C. However, each of the 31 fusants obtained was a mosaic with characteristics derived from both the parents. In the fusion between S. griseus and M. chafcea (Table la), six fusants were similar to
of parental characteristics in seven fusant clones Taxonomic characteristics tested Growth at 40°C NaCl tolerance
Characters of parents -ta -
M. chalcea S. griseus
Number of fusants M. chalcea type S. griseus type Novel type
I 0 0
I 0 0
Utilization of (NH&HP04
3% 5%
.-
6 0 lb
4 3 0
a +, Good growth; ~, no growth. b Nogrowthat 1%. b. Distribution Morphology Characters of parents M. carbonaceae S. griseus
of parental characteristics in 24 fusant clones Taxonomic characteristics tested Growth at 40°C NaCl tolerance -a -
3% 5%
t
24 0 0
1 9 14b
10 14 0
Number of fusants carbonaceae type S. griseus type Novel type a tt, Good growth; - , no growth. b No growth at 0.5%. M.
24 0 0
Utilization of (NH&HP04
-
VOL.
PROTOPLAST
88, 1999 TABLE
2. a. Distribution
FUSION BETWEEN
of carbohydrate utilization
patterns in 7 fusant clones
Sucrose
Mannose
Carbohydrates RafTinose
Ribose
Glycerol
tta -
3
t -
t
-. t-
Characters of parents M. chalcea S. griseus
Number of fusants M. chalcea type S. griseus type
7 0
7 0
145
AND MICROMONOSPORA
STREPTOMYCES
7 0
7 0
7 0
a +, Good growth; - , no growth. b. Distribution
of carbohydrate utilization patterns in 24 fusant clones
Sucrose
Fructose
Mannose
-ta -
-t
-
24 0
13 11
24 0
Characters of parents M. carbonaceae S. griseus
Carbohydrates Xylose
Raffmose
Ribose
Glycerol
tt
-t -
i -t
-
6 18
24 0
23 1
24 0
t
Number of fusants M. carbonaceae S. griseus type
type
a +, Good growth; 5, doubtful growth; -, no growth.
in their tolerance to 3% NaCl, but one was a novel type which showed tolerance to only less than 1% NaCl. Among the 24 fusants obtained by fusion between S. griseus and M. carbonaceae (Table lb), one was similar to M. carbonaceae in its tolerance to 3% NaCI, nine were similar to S. griseus in their tolerance to 5% NaCl and 14 were novel types which showed no growth even in the presence of 0.5% NaCl. Diammoniurn hydrogenphosphate utilization among the 24 fusants was also heterogeneously distributed; 10 were similar to Micromonospora (negative utilization) and 14 were similar to S. griseus (positive utilization). Carbohydrate utilization As shown in Table 2a, all seven fusants between S. griseus and M. chalcea exhibited a carbohydrate utilization pattern similar to that of Micromonospora, none showed the S. griseus pattern. However, in the case of the fusants between S. griseus and M. carbonaceae (Table 2b), the carbohydrate utilizaMicromonospora
TABLE
3.
a. Distribution
tion patterns were distributed heterogeneously among the 24 fusants. Positive utilization of fructose, xylose and ribose (S. griseus type) was detected in 11, 18 and 1, respectively, of the 24 fusants. Utilization of sucrose, mannose, rafhnose and glycerol in all the 24 fusants was similar to that in Micromonospora. Amino acid utilization As shown in Tables 3a and 3b, the seven fusants between S. griseus and M. chalcea heterogeneously inherited the amino acid utilization pattern. Positive utilization of His, Pro, Phe, Hse, Tyr and Cit, similar to S. griseus, was inherited by 2, 7, 1, 5, 7 and 7, respectively, of the seven fusants. Among the 24 fusants between S. griseus and M. carbonaceae, positive utilization of Gly, Ala, His, Val, Pro, Lys, Phe, Hse, Tyr, Cys and Cit, similar to S. griseus; was inherited by 10, 24, 5, 23, 20, 16, 4, 3, 3, 8 and 13 fusants, respectively. In both intergenus fusions, the negative utilization of Try, similar to Micromonospora, was found in all 31
of amino acid utilization
His
Pro
Try
-a tr
f -t
t
patterns in 7 fusant clones
Amino acids Phe Hse
‘W
Cit
3
t
Characters of parents M. chalcea S. griseus
Number of fusants M. chalcea type S. griseus type
5 2
0 7
+t
7 0
h
6 1
2 5
0 7
0 I
a +, Good growth; k, doubtful growth; -, no growth. b. Distribution GUY Characters of parents M. carbonaceae S. griseus
-a -
of amino acid utilization patterns in 24 fusant clones Amino acids LYS Try
Ala
His
Val
Pro
+
3
t tt
k i+
* ++
4 20
8 16
Phe
Hse
Tyr
t-
i-
tt
-
v
tL
t+
24 0
20 4
21 3
21 3
16 8
11 13
Number of fusants 0 19 1 10 24 5 23 a +, Good growth; 5, doubtful growth; -, no growth. M. carbonaceae S. griseus type
type
14
CYS
Cit .-
146
IMADA
ET AL.
f Brosc:1. BIOE’il,. TABLE
__.____
4. a. Distribution
of antibiotic resistance patterns in 7 fusant clones
KM
SM
GM
i 10 -> 150
<5 ,20
12 ,5
4 2 1
1 1 5
Resistance to antibioticsa @g/ml) NA NM DK
LM
KM
IM
Characters of parents M. chalcea S. griseus
Number of fusants M. chalcea type Intermediate type S. griseus type
0 5 2
-2
5
’ 2 ,5
c2 25
:’ 2 x5
-5
0 1 6
3 0 4
6 0 1
7 0 0
5 0 2
a KM, Kanamycin; SM, streptomycin; GM, gentamicin; NA, nearnine; NM, neomycin; DK, dibekacin; LM, lividomycin; IM, istamycin B. Concentration as free base. b. Distribution
Characters of parents M. carbonaceae S. griseus
5
2 i
7 0 0 RM, ribostamycin;
of antibiotic resistance patterns in 24 fusant clones
KM
SM
GM
Resistance to antibiotics” @g/ml) NA NM DK LM
RM
IM
BT -
(10 > 150
<5 .20
c2 >5
<2 ;5
<2 .J5
<’ 2 : 5
‘2 ,5
-1’2 -5
‘; 2 )5
*. 2 ‘, 5
4 19 1
24 0 0
11 10 3
0 3 21
22 0 2
0 2 22
0 18 6
0 10 14
12 12 0
0 0 24
Number of fusants type Intermediate type S. griseus type
M. carbonaceae
a KM, Kanamycin; SM, streptomycin; GM, gentamicin; NA, neamine; NM, neomycin; DK, dibekacin; LM, lividomycin; RM, ribostamycin; IM, istamycin B. Concentration as free base.
fusants. As shown Resistance to aminoglycoside antibiotics in Table 4a, the fusants heterogeneously showed resistance to nine aminoglycoside antibiotics. Resistance to KM (>150/lg/ml), SM (>20), GM (>5), NA (>5), NM (> 5), DK (> 5) and RM (> 5) (S. griseus type) was inherited by 2, 1, 5, 6, 4, 1 and 2, respectively, of the seven fusants, and sensitivity to SM (<5), GM (<2), NM (<2), DK (<2), LM (<2), RM (<5) and IM (<2) (Micromonospora type) was inherited by 4, 1, 3, 6, 7, 5, and 7, respectively, of the seven fusants. A resistance type intermediate between that of the two parents was also observed among the seven fusants; 5, 2, 1 TABLE
5.
a. Distribution of antimicrobial fusant clones B. subtilis
E. coli
spectrum of 7 C. albicans
219
K-12
3147
_i
+-
-
Characters of parents M, chalcea S. griseus
Number of fusants M. chalcea type S. wiseus tvoe a C, Positive antimicrobial tivity. b. Distribution
7 7 7 0 0 0 activity; - , negative antimicrobial
of antimicrobial B. subtilis
219 Characters of parents M. carbonaceae S. griseus
Number of fusants M. carbonaceae S. nriseus type
type
a +, Positive antimicrobial tivity.
ac-
spectrum of 24 fusant clones E. coli
C. albicans
K-12
3147
4
.-
_L
22 2
24 0
24 0
-a
activity; - , negative antimicrobial
ac-
and 1 to KM (lo-150/1g/ml), SM (5-20), GM (2-5) and NA (2-5), respectively. Among the 24 fusants between S. griseus and M. carbonaceae also (Table 4b), the resistance to aminoglycosides was inherited heterogeneously; resistance to KM, GM, NA, NM, DK, LM, RM and BT (S. griseus type) was inherited by 1, 3, 21, 2, 22, 6, 14 and 24 fusants, respectively. An intermediate resistance type between that of the two parents was also observed; 19, 10, 3, 2, 18, 10 and 12 out of 24 fusants showed intermediate type of resistance to KM, GM, NA, DK, LM, RM and IM, respectively, but no intermediate type of resistance to SM (5-20), NM (2-5) nor BT (2-5) was found among the 24 fusants. It was notable that all the 24 fusants resembled Micromonospora in terms of SM resistance but S. griseus in terms of BT resistance. Antibiotic production As shown in Tables 5a and 5b, none of the seven fusants between S. griseus and M. chalcea exhibited antimicrobial activity (Micromonospora type) against any of the tested organisms. Among the 24 fusants between S. griseus and Micromonospora carbonaceae, only two exhibited antimicrobial activity against B. subtilis, however, these two fusants did not show any activity against E. coli. This indicates that the antibiotic is not identical to SM, but is a newly formed antibiotic. DISCUSSION To discover newer substances for medical and agricultural use, new lead compounds are needed and unexplored microorganisms are expected as their source. Accumulated data indicate the probability of the existence of unknown and new microorganisms that remain to be explored in nature. It is also known that new clones are generated by mutations or gene transfer under changing environmental conditions. This knowledge raises the hope of obtaining new bioactive compounds from newly evolved microorganisms in nature. On the other hand, genetic manipulation techniques in known microorganisms, including transformation, transduction and conju-
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PROTOPLAST
FUSION BETWEEN
gation techniques have been developed in laboratories, and could be used to transfer the foreign genes of known clones, or engineered genes to generate novel clones. However, these techniques allow neither recombination between whole genomes nor that with more than two crosses. Therefore, they are inadequate for obtaining new clones for the screening of new bioactive compounds. Under these circumstances, the first successful yield of a new antibiotic-producing clone was reported by our group with interspecies protoplast fusion of streptomycetes (17), although the production ability was unstable. One of the authors, Okanishi et al. (9) reported that the protoplast fusion technique would be promising for obtaining new clones. Using newly constructed methods for improved efficiency of protoplast fusion among streptomycetes, they succeeded in obtaining new clones capable of producing new antibiotics. In this paper, we report the first successful intergenus fusion of actinomycetes and how the parental characteristics are distributed in the fusants. The results showed a fairly good frequency of generation of new clones for the screening of new antibiotic compounds and other characteristics of actinomycetes. In this intergenus experiment, it is of interest to note that almost all fusants between the streptomycete and the two different species of Micromonospora seemed to resemble Micromonospora but not S. griseus in morphological appearance, growth at 40°C and antibiotic productivity. However, most physiological characteristics in each of the 31 fusants did not incline towards any one of the parents but showed mosaic characteristics of both parents. One clone out of the seven fusants between M. chalcea and S. griseus was a novel type and lacked the ability to grow in the presence of 1% NaCl, unlike either of the parents. In the case of fusion between M. carbonaceae and S. griseus, nine out of the 24 fusants were similar to S. griseus in NaCl tolerance (5%) and 14 were a novel type, showing no growth even in the presence of 0.5% NaCl. In regard to carbohydrate utilization, the 24 clones showed heterogeneous utilization patterns of carbohydrates, indicating the generation of new recombinants that were different from both parents. In regard to amino acid utilization, patterns were heterogeneously inherited by the fusants, except that all the fusants obtained exhibited negative utilization of Try (Micromonospora type). Interestingly, in all the intergenus fusants (31 clones), Sue +, Man+, Gly+ and Try-, characteristics specific to Micromonospora, and Ala+ and BT’, as those specific to S. griseus, were expressed. These findings suggest that the former genes link with the raf gene in Micromonospora and the latter with the km gene in S. griseus. The antimicrobial activity of two fusants against B. subtilis was not consistent with that of SM and very stable in spite of three subcultivations. It can be said that the intergenus fusion is a promising technique for the generation of novel clones at a considerably high frequency and is available for efficient screening of new bioactive compounds, although the recombination after the fusion treatment cannot be directed at obtaining certain specified characteristics in the recombinants. On the other hand, in the self-fusion experiments carried out simultaneously, clones showing a stable KM’.Raf+ characteristics have not so far been obtained, whereas the KM’.Raf+ characteristic in the intergenus fusants
STREPTOMYCES
AND MZCROMONOSPORA
147
was very stable, and these fusants were mosaics with characteristics derived from both parents. This suggests that the clones (KM’.Raf+) obtained by intergenus fusion are true recombinants. We are planning to carry out Southern hybridization to verify the recombination as well as to detect DNA fragments expressing each of the characteristics of S. griseus. REFERENCES 1.
Okanishi, M., Suzuki, K., and Umezawa, H.: Formation and
reversionof Streptomyceteprotoplasts: cultural condition and morphological study. J. Gen. Microbial., 80, 389-400 (1976). 2. Shaeffer, P., Cami, B., and Hotchkiss, R. D.: Fusion of bacterial nrotoulasts. Proc. Natl. Acad. Sci., USA, 73. 2151-2155 (1976): 3. Fodor, K. and Alfoldi, L.: Fusion of protoplasts of Bacillus meguterium. Proc. Natl. Acad. Sci., USA, 73, 2147-2150 (1976). 4. Hopwood, D. A., Wright, H. M., and Bibb, M. J.: Genetic recombination through protoplast fusion in Streptomyces. Nature, 268, 171-174 (1977). 5. Urauo, N., Kamimura, M., and Washizu, M.: Physical parameters affecting electrofusion of yeasts: &potential on the surface of yeast protoplasts and osmotic pressure of the solution. J. Biotechnol., 18, 213-224 (1991). 6. Anne, J. and Peberdy, J. F.: Conditions for induced fusion of fungal protoplasts in polyethylene glycol solutions. Arch. Microbial., 105, 201-205 (1975). 7. Godfrey, O., Ford, L., and Hnber, M. L. B.: Interspecies matings of Streptomyces fradiae with Streptomyces bikiniensis mediated by conventional and protoplast fusion techniques. Can. J. Microbial., 24, 994-997 (1978). 8. Ikeda, H., Inoue, M., Tanaka, H., and Omura, S.: Interspecific protoplast fusion among macrolide-producing Streptomycetes. J. Antibiot., 37, 1224-1230 (1984). 9. Okanishi, M., Suzuki, N., and Furuta, T.: Variety of hybrid characters among recombinants obtained by interspecific protoplast fusion in Streptomycetes. Biosci. Biotech. Biochem., 60, 1233-1238 (1996). 10. Okanishi, M., Yamaura, Y., and Furuta, T.: Variety of intracellular products among recombinants obtained by interspecific protoplast fusion in Streptomycetes. Biosci. Biotech. Biochem., 61, 748-751 (1997). 11. Ward, M.,. Davey, M. R., Mathias, R. J. Cocking, E. C., Clothier. R.H.. Balls, M.. and Lucv. J. A.: Effects of aH. Ca*‘, temperature, and protease pretreatment on interkingdom fusion. Somatic Cell Genet., 5, 529-536 (1979). 12. Yamashita, F., Hotta, K., Kurasawa, S,, Okami, Y., and Umezawa, H.: New antibiotic-producing Streptomycetes, selected by antibiotic resistance as a marker. I. New antibiotic production generated by protopfast fusion treatment between Streptomyces griseus and S. tenjimariensis. J. Antibiot., 38, 58-63 (1985). 13. Caso, J. L., Hardisson, C., and Suarez, J. E.: Transfection in Micromonospora spp. Appl. Environ. Microbial., 53, 25442547 (1987). 14. Shirahama, T., Furumai, T., and Okanishi, M.: A modified regeneration method for Streptomycete protoplasts. Agric. Biol. Chem., 45, 1271-1273 (1981). 15. Takada, Y., Kizuka, M., Tanaka, M., and Muto, N.: Establishment of the hostvector system for Micromonospora griseorubida. J. Antibiot., 47. 1167-1170 (1994). 16. Imada, C. and Okamf, Y.: Characteristics of marine actinomycete isolated from deep-sea sediment and production of pglucosidase inhibitor. J. Mar. Biotechnol., 2, 109-l 13 (1995). 17. Hotta, K., Yamashita, F., Okami, Y., and Umezawa, H.: New antibiotic-producing Streptomycetes, selected by antibiotic resistance as a marker. J. Antibiot., 38, 64-69 (1985).
Intergenus Protoplast Fusion between Streptomyces and Micromonospora with Reference to the Distribution of Parental Characteristics in the Fusants CHIAKI IMADA,* MASANORI OKANISHI, AND YOSHIRO OKAMI Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan Received 23 March 1999/Accepted 25 May 1999
The protoplasts of two strains of Mcromonospora which were sensitive to kanamycin (KMS) and utilized raffinose (Raf+), and one strain of Streptomyces griseus which was resistant to KM (KM’) and did not utilize raffinose (Raf-), were prepared, mixed in the presence of polyethylene glycol (PEG) and incubated on regeneration agar plates. Recombinant colonies showing KM’.Raf+ were obtained at a frequency of 2 X 10T6. Their recombinants displayed a signiticant exchange of taxonomic characteristics between the two genera, although the majority appeared similar to the parent Micromonospora in their morphology as well as growth at 4O‘C. Their patterns of utilization of carbohydrates, amino acids and diammonium hydrogenphosphate were different from those of the Micromonospora. Intermediate or novel types which differed from their parents in their tolerance to NaCl and sensitivity to aminoglycoside antibiotics were also observed. Out of the 31 fusants obtained, two showed antimicrobial activity against Bacillus subtilis PC1 219, without any activity against Escherictia coli K-12 or Candida albicans 3147. The active substancemay be a newly formed antibiotic, different from streptomycin in S. griseus. [Key words: protoplast
fusion, Streptomyces,Micromonospora]
aminoethanesulphonic acid (TES) were obtained from Nakalai Tesque Inc. (Kyoto). Lysozyme from chicken egg white and achromopeptidase purified from Achromobatter lyticus were purchased from Wako Pure Chemical Co. (Osaka). All the other chemicals used in the present study were reagent grade. Parental strains M. chalcea IF0 13503 and M. carbonaceae sub sp. aurantiaca KCC-0168 were obtained from The Institute for Fermentation (Osaka), and Kaken Chemical Co. Ltd. (Tokyo), respectively. S. griseus 2C-5 was kindly given by Dr. K. Hotta of National Institute of Infectious Diseases and was maintained in our laboratory. The two former strains were sensitive to KM (2 pg/ml, KM”) and utilized raffinose (Raf+) for growth. S. griseus was resistant to KM (200 fig/ml, KM’) and did not utilize raffinose (Raff). Protoplast fusion The strains employed were cultivated in 100 ml of 172F medium (13) containing 0.2% glycine in a 500 ml Erlenmeyer flask at 27°C on a rotary shaker (200 rpm). The mycelia of these strains at the late logarithmic growth phase (4-6 d) were induced to form protoplasts according to the method of Okanishi et al. (9) with minor modifications. The mycelia obtained from about 10 ml of S. griseus culture were incubated with 0.6 ml of lysozyme solution (40 mg/ml in 0.5 M sucrose), 0.6 ml of achromopeptidase solution (0.2mg/ml in 0.5 M sucrose) and 5 ml of reaction mixture (P3) at 37°C for 3 h, and about 2 X lo9 protoplasts/reaction mixture were obtained. The two strains of Micromonospora were incubated with the same reaction mixture as mentioned above for l-2 d to obtain about 6 x lo9 protoplasts/reaction mixture. The number of protoplasts was counted directly using a Petroff-Hauser bacterial counting chamber under a phase-contrast microscope. Fusion treatment The mixture of protoplasts (approximately 5 X lo* for each) from the two parental strains was centrifuged, and 2ml of PWP medium (14)
Okanishi et al. (1) specified on the culture media, the special conditions required for the preparation of stable protoplasts in actinomycetes, as also on the procedures for regeneration, promoting more protoplast fusion experiments on bacteria (2, 3), actinomycetes (4), yeasts (5), and fungi (6). This has contributed not only to improving the industrially important microbes, but also to a better understanding of the secondary metabolism in antibiotic synthesis. The fusion technique is considered to be a powerful and promising technique for the genetic manipulation of microbes, to induce a high frequency of recombination between different, not only intraspeties but also interspecies microbes (7-lo), and more so between microbes of different kingdoms (11). Subsequently, a new antibiotic obtained by an interspecies cross among Streptomyces was reported by our group (12). The fusion techniques used were greatly improved by Okanishi et al. (9). The recombinants obtained had stable characteristics, and novel antibiotics are expected to be found in recombinants obtained by interspecies protoplast fusion (10). However, no report on the intergenus fusion between different genera of actinomycetes is yet available. This is the first report on protoplast fusion and genetic recombination between one strain of Streptomyces griseus and two strains of different species of Micromonospora. The characteristics of the fusants are described and the distribution of the parental characteristics in the fusants is discussed. MATERIALS
AND METHODS
Chemicals Polyethylene glycol (PEG, molecular weight 1000) and N-Tris (hydroxy methyl) methyl-2* Corresponding author. Present address: Tokyo Univ. Fisheries, 5-7 Konan, Minato-ku, Tokyo 108-8477, Japan. 143
144
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J. BIOXI. BIOENL,
containing 40% PEG 1000 was added to the precipitate, followed by incubation for 3 min at room temperature. The entire contents of the tube were sucked in and out by a Pasteur pipette. The PEG-treated protoplasts were then diluted by the addition of 3 ml of PWP medium. Regeneration after fusion treatment Protoplast regeneration was carried out according to the method of Shirahama et al. (14) using a modified regeneration medium (15). The protoplast suspension obtained by the fusion treatment was serially diluted, plated on MR 0.3s (15) medium for regeneration, and incubated for 14 d at 27-37°C. The colonies developed on the plates were replica-plated onto the selection medium containing 20 pg/ml of KM and 0.5% raffinose in modified SAP-II (6) agar, and incubated for 14 d. SAP-II agar consisted of 900 ml of inorganic solution (0.5 g MgS04.7Hz0, 0.5 ml 10% CaC12.2Hz0 solution, l.Oml TM solution, 1.6 g NH4N03 and 0.5 g K2HP04), 100 ml of 250 mM TES buffer (pH 7.2), and 16g of Agar Noble (Difco). The KM’.Raf+ colonies which appeared were picked and purified three times to remove pseudorecombinants (heteroclones or heterokaryons). They were preserved in SAP-II agar slants containing 20 pg/ml of KM and 0.5% raffinose. Characteristics of the fusant strains The NaCl tolerance of the fusants was tested by the use of SAP-II agar containing 1.0% glucose and varying concentrations of NaCl. Their utilization patterns of carbohydrates and amino acids were determined as follows. Each carbohydrate or amino acid was added to the SAP-II agar in place of glucose or NH4N03 to obtain a final concentration of 1% and 50mM, respectively. The utilization of diammonium hydrogenphosphate (25 mM) in SAP-II agar was also tested in place of NHdN03. Antibiotic resistance was also determined in SAP-II agar containing 1.0% glucose and varying concentrations of the antibiotics employed. Antimicrobial activity The clones were cultured in disposable 24-well plastic plates (Sumitomo Bakelite Co. TABLE
1. a. Distribution
Morphology
Ltd.) on a Minishaker (B. E. Marubishi Co. Ltd., 200 rpm) for 7 d at 27°C according to the method reported previously (16). The medium consisted of 3.Og NaCl, 0.5 g MgS04.7Hz0, 1 ml 10% CaCl* .2Hz0, 1.5 ml TM solution, 0.5 g K2HP04, 15.0 g maltose and 8.0 g Bactosoytone (Difco) in 1 I of deionized water (pH 7.0). Their antimicrobial activities were determined on agar plates seeded with Bacillus subtilis PC1 219, Escherichia coti K- 12 or Candida albicans 3 147. RESULTS Generation of recombinants by protoplast fusion The intergenus fusion treatment, regeneration and replica-plating procedures (described in Materials and Methods section) yielded KM’. Raf+ colonies at a frequency of about 2 x 10 -‘j. The selection medium was SAP-II agar medium containing KM (20 pg/ml) and raffinose (0.5%). In the case of the self-fusion experiments of Micromonospora, KMT. Raf’ colonies were detected as small colonies at almost the same frequency as that following intergenus fusion. However, none of them grew on the selection plate (SKM, +Raf) by subcultivations, indicating that the selected colonies were not true recombinants. In the case of the self-fusion of S. griseus, no KM’.Raf+ colonies were detected. On the other hand, the colonies of KM’.Raf+ obtained by intergenus fusion, did not show any change in the selected characteristics after purification Distribution of parental characteristics in the fusants Morphology, growth at 4O”C, tolerance to NaCl and diammonium hydrogenphosphate utilization In all the experiments of intergenus fusion in this study, almost all fusants were similar to Micromonospora in their morphological appearance as well as their growth ability at 40°C. However, each of the 31 fusants obtained was a mosaic with characteristics derived from both the parents. In the fusion between S. griseus and M. chafcea (Table la), six fusants were similar to
of parental characteristics in seven fusant clones Taxonomic characteristics tested Growth at 40°C NaCl tolerance
Characters of parents -ta -
M. chalcea S. griseus
Number of fusants M. chalcea type S. griseus type Novel type
I 0 0
I 0 0
Utilization of (NH&HP04
3% 5%
.-
6 0 lb
4 3 0
a +, Good growth; ~, no growth. b Nogrowthat 1%. b. Distribution Morphology Characters of parents M. carbonaceae S. griseus
of parental characteristics in 24 fusant clones Taxonomic characteristics tested Growth at 40°C NaCl tolerance -a -
3% 5%
t
24 0 0
1 9 14b
10 14 0
Number of fusants carbonaceae type S. griseus type Novel type a tt, Good growth; - , no growth. b No growth at 0.5%. M.
24 0 0
Utilization of (NH&HP04
-
VOL.
PROTOPLAST
88, 1999 TABLE
2. a. Distribution
FUSION BETWEEN
of carbohydrate utilization
patterns in 7 fusant clones
Sucrose
Mannose
Carbohydrates RafTinose
Ribose
Glycerol
tta -
3
t -
t
-. t-
Characters of parents M. chalcea S. griseus
Number of fusants M. chalcea type S. griseus type
7 0
7 0
145
AND MICROMONOSPORA
STREPTOMYCES
7 0
7 0
7 0
a +, Good growth; - , no growth. b. Distribution
of carbohydrate utilization patterns in 24 fusant clones
Sucrose
Fructose
Mannose
-ta -
-t
-
24 0
13 11
24 0
Characters of parents M. carbonaceae S. griseus
Carbohydrates Xylose
Raffmose
Ribose
Glycerol
tt
-t -
i -t
-
6 18
24 0
23 1
24 0
t
Number of fusants M. carbonaceae S. griseus type
type
a +, Good growth; 5, doubtful growth; -, no growth.
in their tolerance to 3% NaCl, but one was a novel type which showed tolerance to only less than 1% NaCl. Among the 24 fusants obtained by fusion between S. griseus and M. carbonaceae (Table lb), one was similar to M. carbonaceae in its tolerance to 3% NaCI, nine were similar to S. griseus in their tolerance to 5% NaCl and 14 were novel types which showed no growth even in the presence of 0.5% NaCl. Diammoniurn hydrogenphosphate utilization among the 24 fusants was also heterogeneously distributed; 10 were similar to Micromonospora (negative utilization) and 14 were similar to S. griseus (positive utilization). Carbohydrate utilization As shown in Table 2a, all seven fusants between S. griseus and M. chalcea exhibited a carbohydrate utilization pattern similar to that of Micromonospora, none showed the S. griseus pattern. However, in the case of the fusants between S. griseus and M. carbonaceae (Table 2b), the carbohydrate utilizaMicromonospora
TABLE
3.
a. Distribution
tion patterns were distributed heterogeneously among the 24 fusants. Positive utilization of fructose, xylose and ribose (S. griseus type) was detected in 11, 18 and 1, respectively, of the 24 fusants. Utilization of sucrose, mannose, rafhnose and glycerol in all the 24 fusants was similar to that in Micromonospora. Amino acid utilization As shown in Tables 3a and 3b, the seven fusants between S. griseus and M. chalcea heterogeneously inherited the amino acid utilization pattern. Positive utilization of His, Pro, Phe, Hse, Tyr and Cit, similar to S. griseus, was inherited by 2, 7, 1, 5, 7 and 7, respectively, of the seven fusants. Among the 24 fusants between S. griseus and M. carbonaceae, positive utilization of Gly, Ala, His, Val, Pro, Lys, Phe, Hse, Tyr, Cys and Cit, similar to S. griseus; was inherited by 10, 24, 5, 23, 20, 16, 4, 3, 3, 8 and 13 fusants, respectively. In both intergenus fusions, the negative utilization of Try, similar to Micromonospora, was found in all 31
of amino acid utilization
His
Pro
Try
-a tr
f -t
t
patterns in 7 fusant clones
Amino acids Phe Hse
‘W
Cit
3
t
Characters of parents M. chalcea S. griseus
Number of fusants M. chalcea type S. griseus type
5 2
0 7
+t
7 0
h
6 1
2 5
0 7
0 I
a +, Good growth; k, doubtful growth; -, no growth. b. Distribution GUY Characters of parents M. carbonaceae S. griseus
-a -
of amino acid utilization patterns in 24 fusant clones Amino acids LYS Try
Ala
His
Val
Pro
+
3
t tt
k i+
* ++
4 20
8 16
Phe
Hse
Tyr
t-
i-
tt
-
v
tL
t+
24 0
20 4
21 3
21 3
16 8
11 13
Number of fusants 0 19 1 10 24 5 23 a +, Good growth; 5, doubtful growth; -, no growth. M. carbonaceae S. griseus type
type
14
CYS
Cit .-
146
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ET AL.
f Brosc:1. BIOE’il,. TABLE
__.____
4. a. Distribution
of antibiotic resistance patterns in 7 fusant clones
KM
SM
GM
i 10 -> 150
<5 ,20
12 ,5
4 2 1
1 1 5
Resistance to antibioticsa @g/ml) NA NM DK
LM
KM
IM
Characters of parents M. chalcea S. griseus
Number of fusants M. chalcea type Intermediate type S. griseus type
0 5 2
-2
5
’ 2 ,5
c2 25
:’ 2 x5
-5
0 1 6
3 0 4
6 0 1
7 0 0
5 0 2
a KM, Kanamycin; SM, streptomycin; GM, gentamicin; NA, nearnine; NM, neomycin; DK, dibekacin; LM, lividomycin; IM, istamycin B. Concentration as free base. b. Distribution
Characters of parents M. carbonaceae S. griseus
5
2 i
7 0 0 RM, ribostamycin;
of antibiotic resistance patterns in 24 fusant clones
KM
SM
GM
Resistance to antibiotics” @g/ml) NA NM DK LM
RM
IM
BT -
(10 > 150
<5 .20
c2 >5
<2 ;5
<2 .J5
<’ 2 : 5
‘2 ,5
-1’2 -5
‘; 2 )5
*. 2 ‘, 5
4 19 1
24 0 0
11 10 3
0 3 21
22 0 2
0 2 22
0 18 6
0 10 14
12 12 0
0 0 24
Number of fusants type Intermediate type S. griseus type
M. carbonaceae
a KM, Kanamycin; SM, streptomycin; GM, gentamicin; NA, neamine; NM, neomycin; DK, dibekacin; LM, lividomycin; RM, ribostamycin; IM, istamycin B. Concentration as free base.
fusants. As shown Resistance to aminoglycoside antibiotics in Table 4a, the fusants heterogeneously showed resistance to nine aminoglycoside antibiotics. Resistance to KM (>150/lg/ml), SM (>20), GM (>5), NA (>5), NM (> 5), DK (> 5) and RM (> 5) (S. griseus type) was inherited by 2, 1, 5, 6, 4, 1 and 2, respectively, of the seven fusants, and sensitivity to SM (<5), GM (<2), NM (<2), DK (<2), LM (<2), RM (<5) and IM (<2) (Micromonospora type) was inherited by 4, 1, 3, 6, 7, 5, and 7, respectively, of the seven fusants. A resistance type intermediate between that of the two parents was also observed among the seven fusants; 5, 2, 1 TABLE
5.
a. Distribution of antimicrobial fusant clones B. subtilis
E. coli
spectrum of 7 C. albicans
219
K-12
3147
_i
+-
-
Characters of parents M, chalcea S. griseus
Number of fusants M. chalcea type S. wiseus tvoe a C, Positive antimicrobial tivity. b. Distribution
7 7 7 0 0 0 activity; - , negative antimicrobial
of antimicrobial B. subtilis
219 Characters of parents M. carbonaceae S. griseus
Number of fusants M. carbonaceae S. nriseus type
type
a +, Positive antimicrobial tivity.
ac-
spectrum of 24 fusant clones E. coli
C. albicans
K-12
3147
4
.-
_L
22 2
24 0
24 0
-a
activity; - , negative antimicrobial
ac-
and 1 to KM (lo-150/1g/ml), SM (5-20), GM (2-5) and NA (2-5), respectively. Among the 24 fusants between S. griseus and M. carbonaceae also (Table 4b), the resistance to aminoglycosides was inherited heterogeneously; resistance to KM, GM, NA, NM, DK, LM, RM and BT (S. griseus type) was inherited by 1, 3, 21, 2, 22, 6, 14 and 24 fusants, respectively. An intermediate resistance type between that of the two parents was also observed; 19, 10, 3, 2, 18, 10 and 12 out of 24 fusants showed intermediate type of resistance to KM, GM, NA, DK, LM, RM and IM, respectively, but no intermediate type of resistance to SM (5-20), NM (2-5) nor BT (2-5) was found among the 24 fusants. It was notable that all the 24 fusants resembled Micromonospora in terms of SM resistance but S. griseus in terms of BT resistance. Antibiotic production As shown in Tables 5a and 5b, none of the seven fusants between S. griseus and M. chalcea exhibited antimicrobial activity (Micromonospora type) against any of the tested organisms. Among the 24 fusants between S. griseus and Micromonospora carbonaceae, only two exhibited antimicrobial activity against B. subtilis, however, these two fusants did not show any activity against E. coli. This indicates that the antibiotic is not identical to SM, but is a newly formed antibiotic. DISCUSSION To discover newer substances for medical and agricultural use, new lead compounds are needed and unexplored microorganisms are expected as their source. Accumulated data indicate the probability of the existence of unknown and new microorganisms that remain to be explored in nature. It is also known that new clones are generated by mutations or gene transfer under changing environmental conditions. This knowledge raises the hope of obtaining new bioactive compounds from newly evolved microorganisms in nature. On the other hand, genetic manipulation techniques in known microorganisms, including transformation, transduction and conju-
VOL.
88, 1999
PROTOPLAST
FUSION BETWEEN
gation techniques have been developed in laboratories, and could be used to transfer the foreign genes of known clones, or engineered genes to generate novel clones. However, these techniques allow neither recombination between whole genomes nor that with more than two crosses. Therefore, they are inadequate for obtaining new clones for the screening of new bioactive compounds. Under these circumstances, the first successful yield of a new antibiotic-producing clone was reported by our group with interspecies protoplast fusion of streptomycetes (17), although the production ability was unstable. One of the authors, Okanishi et al. (9) reported that the protoplast fusion technique would be promising for obtaining new clones. Using newly constructed methods for improved efficiency of protoplast fusion among streptomycetes, they succeeded in obtaining new clones capable of producing new antibiotics. In this paper, we report the first successful intergenus fusion of actinomycetes and how the parental characteristics are distributed in the fusants. The results showed a fairly good frequency of generation of new clones for the screening of new antibiotic compounds and other characteristics of actinomycetes. In this intergenus experiment, it is of interest to note that almost all fusants between the streptomycete and the two different species of Micromonospora seemed to resemble Micromonospora but not S. griseus in morphological appearance, growth at 40°C and antibiotic productivity. However, most physiological characteristics in each of the 31 fusants did not incline towards any one of the parents but showed mosaic characteristics of both parents. One clone out of the seven fusants between M. chalcea and S. griseus was a novel type and lacked the ability to grow in the presence of 1% NaCl, unlike either of the parents. In the case of fusion between M. carbonaceae and S. griseus, nine out of the 24 fusants were similar to S. griseus in NaCl tolerance (5%) and 14 were a novel type, showing no growth even in the presence of 0.5% NaCl. In regard to carbohydrate utilization, the 24 clones showed heterogeneous utilization patterns of carbohydrates, indicating the generation of new recombinants that were different from both parents. In regard to amino acid utilization, patterns were heterogeneously inherited by the fusants, except that all the fusants obtained exhibited negative utilization of Try (Micromonospora type). Interestingly, in all the intergenus fusants (31 clones), Sue +, Man+, Gly+ and Try-, characteristics specific to Micromonospora, and Ala+ and BT’, as those specific to S. griseus, were expressed. These findings suggest that the former genes link with the raf gene in Micromonospora and the latter with the km gene in S. griseus. The antimicrobial activity of two fusants against B. subtilis was not consistent with that of SM and very stable in spite of three subcultivations. It can be said that the intergenus fusion is a promising technique for the generation of novel clones at a considerably high frequency and is available for efficient screening of new bioactive compounds, although the recombination after the fusion treatment cannot be directed at obtaining certain specified characteristics in the recombinants. On the other hand, in the self-fusion experiments carried out simultaneously, clones showing a stable KM’.Raf+ characteristics have not so far been obtained, whereas the KM’.Raf+ characteristic in the intergenus fusants
STREPTOMYCES
AND MZCROMONOSPORA
147
was very stable, and these fusants were mosaics with characteristics derived from both parents. This suggests that the clones (KM’.Raf+) obtained by intergenus fusion are true recombinants. We are planning to carry out Southern hybridization to verify the recombination as well as to detect DNA fragments expressing each of the characteristics of S. griseus. REFERENCES 1.
Okanishi, M., Suzuki, K., and Umezawa, H.: Formation and
reversionof Streptomyceteprotoplasts: cultural condition and morphological study. J. Gen. Microbial., 80, 389-400 (1976). 2. Shaeffer, P., Cami, B., and Hotchkiss, R. D.: Fusion of bacterial nrotoulasts. Proc. Natl. Acad. Sci., USA, 73. 2151-2155 (1976): 3. Fodor, K. and Alfoldi, L.: Fusion of protoplasts of Bacillus meguterium. Proc. Natl. Acad. Sci., USA, 73, 2147-2150 (1976). 4. Hopwood, D. A., Wright, H. M., and Bibb, M. J.: Genetic recombination through protoplast fusion in Streptomyces. Nature, 268, 171-174 (1977). 5. Urauo, N., Kamimura, M., and Washizu, M.: Physical parameters affecting electrofusion of yeasts: &potential on the surface of yeast protoplasts and osmotic pressure of the solution. J. Biotechnol., 18, 213-224 (1991). 6. Anne, J. and Peberdy, J. F.: Conditions for induced fusion of fungal protoplasts in polyethylene glycol solutions. Arch. Microbial., 105, 201-205 (1975). 7. Godfrey, O., Ford, L., and Hnber, M. L. B.: Interspecies matings of Streptomyces fradiae with Streptomyces bikiniensis mediated by conventional and protoplast fusion techniques. Can. J. Microbial., 24, 994-997 (1978). 8. Ikeda, H., Inoue, M., Tanaka, H., and Omura, S.: Interspecific protoplast fusion among macrolide-producing Streptomycetes. J. Antibiot., 37, 1224-1230 (1984). 9. Okanishi, M., Suzuki, N., and Furuta, T.: Variety of hybrid characters among recombinants obtained by interspecific protoplast fusion in Streptomycetes. Biosci. Biotech. Biochem., 60, 1233-1238 (1996). 10. Okanishi, M., Yamaura, Y., and Furuta, T.: Variety of intracellular products among recombinants obtained by interspecific protoplast fusion in Streptomycetes. Biosci. Biotech. Biochem., 61, 748-751 (1997). 11. Ward, M.,. Davey, M. R., Mathias, R. J. Cocking, E. C., Clothier. R.H.. Balls, M.. and Lucv. J. A.: Effects of aH. Ca*‘, temperature, and protease pretreatment on interkingdom fusion. Somatic Cell Genet., 5, 529-536 (1979). 12. Yamashita, F., Hotta, K., Kurasawa, S,, Okami, Y., and Umezawa, H.: New antibiotic-producing Streptomycetes, selected by antibiotic resistance as a marker. I. New antibiotic production generated by protopfast fusion treatment between Streptomyces griseus and S. tenjimariensis. J. Antibiot., 38, 58-63 (1985). 13. Caso, J. L., Hardisson, C., and Suarez, J. E.: Transfection in Micromonospora spp. Appl. Environ. Microbial., 53, 25442547 (1987). 14. Shirahama, T., Furumai, T., and Okanishi, M.: A modified regeneration method for Streptomycete protoplasts. Agric. Biol. Chem., 45, 1271-1273 (1981). 15. Takada, Y., Kizuka, M., Tanaka, M., and Muto, N.: Establishment of the hostvector system for Micromonospora griseorubida. J. Antibiot., 47. 1167-1170 (1994). 16. Imada, C. and Okamf, Y.: Characteristics of marine actinomycete isolated from deep-sea sediment and production of pglucosidase inhibitor. J. Mar. Biotechnol., 2, 109-l 13 (1995). 17. Hotta, K., Yamashita, F., Okami, Y., and Umezawa, H.: New antibiotic-producing Streptomycetes, selected by antibiotic resistance as a marker. J. Antibiot., 38, 64-69 (1985).