Agric. Biol. Chem., 49 (1), 133-140, 1985 133

Fusion of Spheroplasts and Genetic Recombination of Zymomonasmobilis Hideshi Yanase, Masaru Yasui, Takayuki Miyazaki and KenzoTonomura Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Mozu-ume-machi, Sakai, Osaka 591, Japan Received July 1.8, 1984

Spheroplasts of Zymomonasmobilis (Z-6) were prepared by cultivating it in a hypertonic mediumcontaining penicillin G or glycine at 30°C for more than 6 hr. Thereversion of spheroplasts to a bacillary form was observed at frequencies of 10~2 to 10~3 whenspheroplasts were incubated on hypertonic solid agar plates overlaid with soft agar. Spheroplast fusion was attempted with polyethylene glycol 6000 by crossing two mutants lacking the ability to ferment sugars. Fusants were selected, and obtained at high frequencies. The properties of a stable fusant are discussed.

Recently broad host-range plasmids have Media. For the cultivation of Zymomonasstrains, RM and T media were used. RMmediumcontained 2% been transferred into Zymomonasmobilis by glucose, 1.0% yeast extract and 0.2% KH2PO4, pH 6.0. T conjugation.1 ~4) However, transformation medium contained 10% glucose, 1% yeast extract, 1% and transduction have not yet been accom- KH2PO4, 1% (NH4)2SO4 and 0.05% MgSO4-7H2O, pH plished. Besides, cell fusion could be regarded 5.6. For the formation of spheroplasts, S mediumwas used as a useful method for strain improvement. containing 20%sucrose, 2%glucose, 1%yeast extract, There have been manyreports indicating pro- 0.2% KH2PO4 and 0.2% MgSO4à"7H2O, pH 6.0. For the toplast fusion in Gram-positive bacteria such regeneration, R mediumwas used containing 16% sor- as Bacillus^ Staphylococcus,6) Brevibacte- bitol, 2% glucose, 1% yeast extract and 0.2% KH2PO4, pH riurn1) and Streptomyces^ but only a few on Gram-negative bacteria; for instance, Coetzee Spheroplast formation. An aliquot (0.1 ml) of a culture et al.9) have reported intraspecific fusion in of a Zymomonasstrain was inoculated into 5ml of T medium and cultured for 12hr at 30°C. The culture at the Providence alcalifaciens, and Rastogi et al.10) late exponential phase (5 x 108 cells/ml) was diluted 1 : 4 in Mycobacterium aurum. Therefore, we de- 6.0.with S medium containing 3.8% glycine or 250 units/ml of cided to attempt spheroplast fusion in Z. penicillin G. After 6 to 12hr incubation, almost all of the mobilis, and obtained fusants at high frequen- vegetative cells had been converted to a spherical form. cies. This paper describes the procedure for The total numberof cells in the spheroplast mixture was spheroplast formation, spheroplast fusion, and determined with a haemocytometerunder a microscope. cell wall regeneration of Z. mobilis for the first Table I. Strains of ZymomonasUsed time. _ T ^ , Fermentationofstrain Invertase a-Gal _ c oRaf Sue Era MATERIALS AND METHODS Z -6 W In d u c ib le Bacterial strains. These are listed in Table I. Zymomonas Z -6 -C M C o n stitu tiv e - mobilis subsp. mobilis IFO 13756 (Z-6) is a wild strain. The Z - 6 - C f r i T M C o n s t i t u t i v e - Z-6-C strain is a spontaneous mutant derived from Z-6, Z - 6 - C s u e " M C o n s t i t u t i v e - and produces invertase constitutively. Z-6-C fru ~ and Z-6- C sue" are mutants derived from Z-6-C by TV-methyl-JV'- Z-6, Zymomonas mobilis subsp. mobilis IFO13756; nitro-TV-nitrosoguanidine (NTG) treatment, which have Raf, raffinose; Sue, sucrose; Fru, fructose; a-Gal, a- lost the ability to ferment fructose or sucrose, respectively. galactosidase; W, wild; M, mutant. 134 H. Yanase et al.

The numbers of osmotic resistant cells in the spheroplast Electron microscopy of spheroplasts. Spheroplasts were mixture were determined as follows. The spheroplast fixed by a modification of the method of Sagara et al.ll) mixture was diluted with 0.85% saline solution to expose This involved treatment with 3%(v/v) glutaraldehyde in the cells to low osmotic pressure, and spread onto an agar phosphate buffer (pH 7.0) containing 1 m sucrose followed plate of RMmedium. The numbers of colonies which by post-fixation in 1 %(w/v) OsO4 for 4hr; both steps were appeared were determined after 5 days incubation at 30°C, performed at 4°C. The fixed material was dehydrated in a and defined as osmotic resistant cells. The number of graded ethanol dilution series and embeddedin Epon 812. spheroplasts in the spheroplast mixture was calculated by This sections were prepared with a LKBultramicrotome deducting the number of osmotic resistant cells from the with glass knives, and viewed under a Hitachi H-300 number of cells. The frequency of spheroplast formation Electron Microscope. wasexpressed as the ratio of the numberof spheroplasts to the total numberof cells. Chemicals. Penicillin G was purchased from Meiji Seika Co., Ltd. (Tokyo). DNase I was obtained from Regeneration of spheroplasts. The spheroplast mixtures Sigma Chemical Co. (St. Louis). All other reagents were were diluted with R medium and plated on R medium of the highest grade available from commercial sources. containing 1.5% agar (R-solid medium). R medium con- taining 0.8% agar (R-soft medium) was overlaid onto the RESULTS surface of the R-solid medium.The numbers of colonies were determined after 3 to 5 days incubation at 30°C. The number of regenerated cells was calculated by deducting SpheroplastIn general,formationgram-negative bacteria are the numberof osmoicresistant cells fromthe numberof colonies on R medium. The frequency of regeneration was known to be converted into spherical bodies expressed as the ratio of the numberof regenerated cells to by a lysozyme-EDTA method.12) However, the numberof spheroplasts. spheroplasts of Z. mobilis (Z-6) could not be formed with this method. Since it is known Spheroplast fusion. Spheroplasts of two strains, Z-6-C fru~ and Z-6-C sue", were mixed and harvested by that the biosynthesis of bacterial cell walls is specifically inhibited by penicillins or gly- centrifugation at 3000rpm for lOmin at 4°C. They were washed with 10mM Tris-HCl, pH 7.2, containing 50mM cine,13) the organism was cultured in S medium CaCl2 and 20% sucrose. The washed spheroplasts were containing penicillin G or glycine to generate resuspended in a one-tenth volume of 10 mMTris-HCl, pH 7.2, containing 50mM CaCl2 and 33% polyethylene glycol spheroplasts; spherical cells were observed un- 6000 (PEG 6000). After incubation at 30°C for 60 min, the der a microscope. Spherical bodies began to suspension was diluted with Raffinose-R medium appear after 3 hr cultivation, and more than (R-medium containing 5%raffinose instead of glucose). 99.99% of the cells had been converted to a For the selection of fusants whose ability to ferment raffinose had been restored, Raffinose-R medium was 100r i n n n n n used. The suspension was spread on a 1.5% agar plate of Raffinose-R medium, and overlaid with the same medium containing 0.8% agar. After 14 days incubation at 30°C, colonies were isolated and fusants were examined as to their sugar fermentation properties. Analyses. Ethanol was quantitatively determined with a Hitachi Gas Chromatograph Model 163 equipped with a flame ionizing detector and a stainless steel column 0 100 200 00 0 1 0 0 D (3.2mmx2m) packed with Porapak Q (80 to 100mesh). The conditions for analysis were as follows: Carrier gas, Fig. 1. Effects of Concentrations of Penicillin G and nitrogen (40ml/min); temperature, 160~ 185°C for the Glycine on Spheroplast Formation of Z. mobilis Z-6. column, 230°C for the injection and detector; internal Spheroplasts of Z-6 were prepared by cultivation in S standard, isopropanol. mediumcontaining penicillin G and glycine at the con- Glucose, fructose, sucrose, melibiose and raffinose were centrations indicated at 30°C for lOhr. The numbers of analyzed and determined with a Liquid Chromatograph spheroplasts and osmotic resistant cells were calculated by equipped with a Shodex RI Model SE-ll and an Ionpak the method described in Materials and Methods. jii^ , KS-802 column. The conditions for analysis were as ratio of the numberof spheroplasts to the numberof total follows: Effluent, H2O(l.O ml/min); column temperature, 60°C. cells, percent; $p , ratio of the number of osmotic resistent cells to the number of total cells, percent. Spheroplast Fusion of Zymomonas 135

Fig. 2. Electron Micrograph of a Thin Section of Penicillin G-Induced Spheroplats of Z. mobilis Z-6 spherical form after 10hr. Figure 1 shows the effects of the concentrations of penicillin G and glycine, respectively, on the formation of spheroplasts of Z. mobilis. The optimum con- centrations were determined to be 100 units/ml for penicillin G and 3% for glycine, since the ratio of the number of osmotic resistant cells to the number of spheroplasts was lowest at that concentration. An electron micrograph of a thin section of a spheroplast of Z. mobilis is shown in Fig. 2. 1.0% Detached cell walls and exposed cytoplasmic 3.0%

5.0% (%) membraneswere observed on spheroplasts. 50100 Frequency of -, regeneration P mmmmtm^m fmmmmmmm |Wa:ffl=;¥ffl$S?;-ft-:-;-;-K-;-;-K-3Kxmxmx-mK^m l.ixlO"2 Regeneration and colony formation of Fig. 3. Effects of Concentrations of Penicillin G or spherop lasts Glycine on Regeneration of Z. mobilis Z-6. For the regeneration of spheroplasts of Spheroplasts of Z-6 were prepared by cultivation in S yeast and gram-positive bacteria, an osmotic mediumcontaining the indicated concentrations of penic- stabilizer such as 0.6n KC1,14) 0.5n NaCl,15) illin G or glycine. The spheroplasts were regenerated in R medium.After 3 to 4 days incubation, the numbers of 0.5n sodium succinate,7) 20% sucrose16) or colonies were determined. Xv, ratio of the numbers of 16%sorbitol17) has been used. Therefore, the regenerated cells to the number of colonies on R medium; effect of these stabilizers on the regeneration of mi , ratio of the numbers of osmotic resistant cells to the Z. mobilis was investigated; a stabilizer was numbers of colonies on R medium. added to R medium. Amongthem, sucrose and sorbitol were found to be most suitable. basal medium. After 3 to 5 days incubation, As sorbitol is not utilized by Z. mobilis and colonies were formed on R medium.The re- also does not inhibit its growth, 16% sorbitol generation frequency is generally affected by was added to the regeneration mediumas an various factors. The effects of the concen- osmotic stabilizer. To regenerate vegetative trations of penicillin G and glycine added at cells from Z-6 spheroplasts, spheroplasts were the time of spheroplast formation on the re- spread onto R solid medium and then R soft generation of spheroplasts were investigated. mediumwas overlaid onto the surface of a As shown in Fig. 3, spheroplasts prepared with 136 H. Yanase et al lower concentrations of penicillin G or glycine to the regeneration medium.16) However, no showed higher frequencies of regeneration, but effect of such plasma expanders was observed under those conditions, that is, 50 units/ml of in the regeneration of Z-6 spheroplasts. penicillin or 0.5% glycine, about half of the colonies which appeared on R mediumwere derived from osmotic resistant cells. ExperimentalTo examinesystemintraspecificof spheroplastfusion, fusiona genetic Consequently, the optimum concentrations marker such as auxotrophy is necessary. were determined to be 200 units/ml for penicil- However,the isolation of auxotrophic mutants lin G and 3% for glycine, since at those of Z. mobilis was difficult, since the growth ofa concentrations higher regeneration frequencies wild strain became poor in minimummedia and lower ratios of osmotic resistant cells to after repeated transfers, even when regenerated cells were obtained. Skotonicki's minimummedium18) was used. Recently, it has been reported that the re- Therefore, we used mutants that had lost the generation frequency of protoplasts in Bacillus ability to ferment sucrose (Z-6-C sue") or subtilis is raised by the addition of an artificial fructose (Z-6-C fru"). plasma expander such as 3%polyvinyl pyr- The wild strain, Z. mobilis (Z-6), fermented rolidone, 3%vinyl pyrrolidone or 3%dextran, glucose, fructose and sucrose to produce

(a) ,. (b) .(c) (d) S 0.5-;' à" à"L , -5-E

4J / ' l t d ' S 12rl0-4'i/ å ' / t/ \ ! ~^s £0.3A' -\ / \ / -\ ,; -3a=

1]'^°-2"AD^'a""a""D å A d-å¡-a -y'A å¡-d-a à" y "2|o

°°° °0122436480 12243648012243648012243648°"*" Cultivation (hr) Fig. 4. Course of Fermentation on Various Sugars of the Z-6-C Strain. Z-6-C was cultured in RMmedium containing 2%glucose (a), 2% fructose (b), 2%sucrose (c) or 5% raffinose (d). Sugars were determined by liquid chromatography and ethanol by gas chromatography. Growth of the organism was determined by measuring the absorption at 610nm. Growth ( ), glucose (#), fructose (A), sucrose (å ), raffinose (O), melibiose (A), ethanol (å¡)à"

(a) (b) (c) (d)

S 0.5- ;--... å å å å 5"

|2 r^0.4o / ,-a-a-a_a,, K "4=^ ^ 20.3\/ à" \ \ "3-^|

101 -O0.2- ll O-D-D ' -\J.A A A à" VA-^A"^A-2^^

*ZS T3I A/ / A k/ JW a'y^v>V«A--A _ ^oS- C

å § L0 ^X. \ L- j^-^u å å ^å "à"å -.-:I £2 1/70 0 122436480 122436 480 122436480 1224 3648° Cultivation (hr) Fig. 5. Course ofFermentation on Various Sugars of the Z-6-C fru Strain. The experimental conditions were the same as in Fig. 4. Growth ( ), glucose (#), fructose (A), sucrose (å ), raffinose (O), melibiose (A), ethanol (å¡)à" Spheroplast Fusion of Zymomonas 137

(a) m (b) (c) (d) S à"' c ^ q.5- ; à" à" å -5à"-

å o2 r- 0.4v / '^A ii-å -å -å -å £-°-o-o-o_ 4 |g

« *° V \# ll g130.2à" Aa-å¡-å¡ - Vd-d-a" à" -2a>cu

£ *o.iå :X å M - - -itic iol*oUy\,..W/\ . ,1..,- J Iq^ 0 12 24 36 480 12 24 36 480 12 24 36 480 12 24 36 48 Cultivation (hr) Fig. 6. Course ofFermentation on Various Sugars of the Z-6-C sue strain. The experimental conditions were the same as in Fig. 4. Growth ( ), glucose (0), fructose (A), sucrose (å ), raffinose (O), melibiose (A), ethanol (å¡)à"

Table II. Frequency of Spheroplast Fusion between Z-6-C fru Z-6-C sue"

C rossO Frequency of apparent T reatm en t Frequency of fn T regen eration recom bin an ts5

9 x lO7 33 % PE G 2 x lO ' < 1(T 2 x lO8 33 % PE G 2 x lO " < 10 ~ 9 x lO7 2 x lO8 33 % P EG 4 x lO " x lO " 9 x lO7 2 x lO8 33 % P E G 4 x lO " 1 x lO " + D N asec

a The numbers shows cell counts in ml. b Ratio of the number of colonies on the selection plate to the number of spheroplasts in the mixture prior to fusion. c In the presence of5/ig/ml ofDNase I. ethanol, but not raffinose. Z-6-C derived from could ferment neither sucrose nor raffinose, as the wild strain produced invertase consti- shown in Fig. 6. The fermentation of sucrose tutively. Figure 4 illustrates fermentations on appears to involve both sucrose permease and glucose, fructose, sucrose and raffinose by the invertase. As invertase was found in the sue" strain. It acquired the ability to ferment raf- strain, the deficiency of sucrose fermentation finose, compared with the wild strain, but seemed to be caused by a deficiency of sucrose melibiose remained in the culture medium permease. Morever, assuming that the sucrose unhydrolyzed (Fig. 4-d). Therefore, it was permeasemaybe responsible for the uptake of assumed that Z-6-C is able to take in raffinose raffinose, it would be more understandable and convert it to melibiose and fructose with that the strain had lost the ability to ferment constitutively formed invertase, and the fruc- raffinose as well as sucrose. tose formed is fermented to ethanol. No a- galactosidase activity was found in this organism. BothSpheroplaststrains, fusionZ-6-C fru" and Z-6-C sue", Characteristics of sugar fermentation by the were converted to spheroplasts. They were Z-6-C fru~ strain are shown in Fig. 5. It is mixed, suspended in 33% PEGsolution and evident that fructose fermentation was de- plated on a selection plate (Raffinose-R me- ficient in the strain. The Z-6-C sue" strain dium). After incubation at 30°C for 10 to 15 138 H. Yanase et al.

(a) (b) (c) (d)

~ 0.5à" à" " I- -5- *å * - - > ?\ S -g 2r^i" 0.4 à" ;* å -±-a*^ '' / Q »-' 4 3&s '£ c \ ; a V / \; o" ;=°-3\/ å \i å U-^-3M

1 O«« \,' T/ \l fOE

la 5 A/ /*\/ '/\ "^° +5 i0.1à" /A å à"/X - If\ -1££ ^oL o L^r V^ -i-. å Jjgs^toi,^g^ J0^g 0 12 2436 48 0 12 24 36 480 12 24 36480 12 24 364.8 Cultivation (hr) Fig. 7. Course ofFermentation on Various Sugars of a Fusant (Z-6-C fru" x Z-6-C sue"). The experimental conditions were the same as in Fig. 4. Growth ( ), glucose (#), fructose (A), sucrose (å ), raffinose (O), melibiose (A), ethanol (å¡)à" days, colonies appeared on the plate. As a those in Z-6-C (Fig. 4). However, fructose control, a parental strain was treated by the fermentation could be observed for the fruc- same procedure as above, but no colonies tose intracellularly produced from raffinose. appeared. The results of the spheroplast fusion Accordingly, internal fructose appeared to be are shown in Table II. Frequencies of the metabolized by a gene product of the sue" fusion were expressed in terms of the ratio of parent in the fusant, but fructose uptake was the number of prototrophic colonies which not completely compensated for by the parent. appeared on the selection plate to the total Almost all of the fusants obtained showed the number of spheroplasts. Judging from the frequencies of regeneration of 10~3 for the sameproperties. spheroplasts of Z-6-C fru~ and Z-6-C sue", DISCUSSION the frequency offusion of 10~4 would be much higher than would be expected. Such a high The spheroplasts of Z. mobilis mutants, Z-6- frequency was assumed to be due to some C fru" and Z-6-C sue", were mixed together other phenomenon, as will be discussed later. and fused. Prototrophic colonies were ob- The addition of deoxyribonuclease I to the served at a high frequency of 10~4 of total fusion mixture prior to the treatment with spheroplasts. Judging from the frequencies of PEG 6000 had no effect on the frequency of regeneration of 10~3, this seemedto be rather fusion, as shown in Table II, indicating that high. To examine whether the prototrophic the recombinants are probably not caused by colonies are true fusants or not, the following transformation with free DNA from lysed experiment was conducted as a control. cells. In order to isolate a stable recombinant, Approximately 109 vegetative cells/ml of the a number of colonies on the selection plate two strains were mixed, spread onto Raffinose- were transferred to Raffinose-RM medium, R medium, and cultured for 10 to 14 days; very and cultured on it repeatedly. During this small colonies appeared. This phenomenon procedure, approximately one-tenth of the seemed to be a result of the growth of the Z-6- prototrophic colonies retained the ability to C sue" strain in the presence of the Z-6-C fru" ferment raffinose stably, but the rest lost it. strain; as shown in Fig. 5, the Z-6-C fru~ The properties of a prototrophic recom- strain takes in raffinose and degrades it to form binant (fusant) were investigated. As shown in fructose and melibiose, which are not metabo- Fig. 7, the fusant had completely regained the lized further but are secreted into the medium. sameability of raffinose fermentation but not Using the fructose, Z-6-C sue" could grow on that of fructose fermentation, comparedwith the plate. Accordingly, when these colonies Spheroplast Fusion of Zymomonas 139

Table III. Genetic and Functional Properties of Mutants and a Fusant

G e n e F er m e nt a t io n of

S tra in fr uA fru B su c A su cB F ru S u e R a f

Z -6 -C + 4 - + . + + + + Z -6 - C f ri T + + + Z -6 - C s u e + + + + F u sa n t ( + ) + + + + + +

fruA; transport of fructose; fruB; utilization of fructose; sucA; sucrose permease; sucB; invertase.

were cultured in RMmediumcontaining 5% imperfect during cell fusion. raffinose instead of glucose at 30°C for 1 to 2 Z. mobilis is a potentially useful organism weeks, no growth was observed. In order to for the industrial production of ethanol. This isolate a true fusant, the prototrophic colonies paper has indicated the possibility of genetic were cultured in Raffinose-RM medium re- manipulation of Z. mobilis through sphero- peatedly. During this procedure, approxi- plast fusion. As a host-vector system as well mately one-tenth of the prototrophic col- as transformation in Z. mobilis has not yet onies tested retained the ability to ferment been established, spheroplast fusion seems to raffinosestably. This indicates that the fre- be the most useful at present. Further genetic quency of spheroplast fusion was approxi- improvement of Zymomonasstrains by sphe- mately 10~5 of the total spheroplasts. roplast fusion are nowin progress. As shown in Fig. 7, it is evident that the fusant has regained the ability to ferment Acknowledgment.This work was supported in part by a Grant-in-Aid for Scientific Research from the raffinose. However, the fusant only grew Ministry of Education, Science and Culture of Japan. slightly on a medium containing fructose. The characteristics of the fusant are summa- REFERENCES rized in Table III. The genes involved in fruc- tose transport and fructose utilization were 1) M. L. Skotnicki, D. E. Tribe and P. L. Rogers, Appl. Environ. MicrobioL, 40, 7 (1980). designated as fruA and fruB, respectively. 2) K. Tonomura, N. Kurose, S. Konishi and H. The genes involved for sucrose permease and Kawasaki, Agric. Biol. Chem., 46, 2851 (1982). invertase were designated as sucAand sucB, 3) E. L. Dally, H. W. Stokes and D. E. Eveleigh, respectively. From the results described Biotechnol. Lett., 4, 91 (1982). 4) V. C. Carey, S. K. Walia and L. O. Ingram, Appl. above, the genotypes of the Z-6-C, Z-6-C fru" , Environ. MicrobioL, 46, 1 163 (1983). and Z-6-C sue" strains are expressed as fol- 5) K. Foder and L. Alfoldi, Proc. Natl. Acad. Sci. lows: Z-6-C,fruA+ fruB+ sucA+ sucB+; Z-6- U.S.A., 73, 2147 (1976). C fm~,fruA~ fruB~ sucA+ sucB+; and Z-6- 6) F. Gotz, S. Ahrne and M. Lindberb, /. Bacteriol, C sue" ,fruA+ fruB+ sucA~ sucB+. It was as- 145, 74 (1981). 7) H. Kaneko and K. Sakaguchi, Agric. Biol. Chem., 43, sumed that in Z-6-C, raffinose was incorpo- 1007 (1979). rated into cells by the sucrose permease of 8) D.A. Hopwood,H. M. Wright, M. J. BibbandS. N. sueA, the incorporated raffinose was degrad- Cohen, Nature (London), 268, 171 (1977). ed by the invertase of sucB, and the fructose 9) J. N. Coetzee, F. A. Sirgel and G. Lecatsas, J. Gen. formed was utilized by thefruB gene product. MicrobioL, 114, 313 (1979). The fusant became able to ferment raffinose 10) N. Rastogi, H. L. David and E. Rafidinarivo, /. Gen. MicrobioL, 129, 1227 (1983). on crossing offruA" fruB~ sucA+ sucB+ and ll) Y. Sagara, K. Fukui, F. Ota, N. Yoshida, T. fruA+ fruB+ sucA~ sucB+. However, the Kashiyama and M. Fujimoto, Jpn. J. MicrobioL, 15, growth on fructose was still low. According- 73 (1971). ly, it appeared that the recombination was 12) T. Miura and S. Mizushima, Biochim. Biophys. Acta, 140 H. Yanase et al.

193, 268 (1969). Microbiol, 80, 389 (1974). J. L. Strominger, K. Izaki, M. Matsuhashi and D. J. T. Akamatsu and J. Sekiguchi, Agric. Biol. Chem., Tipper, Fed. Proc, Fed. Am. Soc. Exp. BioL, 26, 9 45, 2887 (1981). (1967). P. van Solingen and J. B. van Der Plaat, /. BacterioL, 130, 946 (1977). L. Ferenczy and A. Maraz, Nature (London), 268, 524 (1977). A. E. Goodman, P. L. Rogers and M. L. Skotnicki, M. Okanishi, K. Suzuki and H. Umezawa, /. Gen. Appl. Environ. Microbiol., 44, 496 (1982).