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Factors affecting protoplast formation and regeneration in three strains of Streptoverticillium
JOURNALOF IIICROBIOLOGICAL IETHODS ELSEVIER
Journal of Microbiological Methods 19 (1994) 197-206
Factors affecting protoplast formation and regeneration in three strains of Streptoverticillium A n n - K a r o l i n Scheu, J u a n Soliveri *, M a r i a - E n r i q u e t a A r i a s Departamento de Microbiologia y Parasitologia. Universidadde Alcal6 de Henares, 28871-Alcal~ de Henares, Madrid, Spain
Received 25 May 1993; revision received 2 October 1993; accepted 5 October 1993
Abstract
Conditions are reported for efficient protoplast formation and regeneration in three strains of Streptoverticillium. Mid-exponential phase mycelia, grown in Tryptic-soy broth supplemented with 1.5% (w/v) glycine, were highly sensitive to lysozyme, but the protoplasts did not regenerate. The presence of Ca ÷2 in protoplast formation media was required for cell regeneration. Mg +2 improved the regeneration yield only in the case of Streptoverticillium griseocarneum NCIMB 40447. Protoplasts only regenerated in media containing complex nitrogen and carbon sources. Maximum cell regeneration was achieved with protoplasts obtained from mid-exponential growth phase mycelia and plated on an undehydrated Bennet agar overlayered with the same medium containing a lower agar concentration. Sucrose proved to be the optimal osmotic stabilizer and was added to the media for protoplast formation and regeneration. Key words: Streptoverticillium; Protoplast production; Protoplast regeneration
I. Introduction
One of the basic techniques for D N A cloning in actinomycetes is protoplast preparation for the efficient regeneration of normal mycelia to carry out protoplast transformation or fusion and so bypass the barriers encountered with other methods. The development of these techniques for Streptomyces species [1] has considerably enhanced the potential for the genetic manipulation of this industrially important genus. Although Streptoverticillium strains can produce several antibiotics [2], ~ponding
author. Tel: 34.1.8854693. Fax: 34.1.8854660. Email: [email protected]
0167-7012/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 7 0 1 2 ( 9 3 ) E 0 0 6 5 - Z
198
A.-K. Scheuet al./Journalof MicrobiologicalMethods 19 (1994) 197-206
no investigations have addressed protoplast formation. In this sense, and in order to compare the production and regeneration of protoplasts in both genera, classical procedures described for Streptomyces have been tested in three species of Streptoverticillium. The two very closely related genera, Streptomyces and Streptoverticillium, show differences in morphology and physiology [2,3]. In this report, efficient reliable methods for protoplast formation and regeneration in Streptoverticillium strains, based on techniques described for Streptomyces, have been developed. Some differences among factors affecting these processes with either genera have been also established.
2. Materials and methods
2.1. Bacterial strains, mycelial growth and glycine effect In this study three antibiotic producing strains were used: Streptoverticillium griseocarneum NCIMB 40447, an isolate from Spanish soil [4,5,6], Streptoverticillium ladakanum CECT 3263 and Streptoverticillium netropsis CECT 3265. These strains share a high phenotypic similarity and will be referred to hereafter as strain 40447, 3263 and 3265, respectively. The strains were stock-cultured on a modified Bennet agar (BA) medium [3] which contained 10 g glucose (Merck), 1 g Lab-Lenko powder (Oxoid), 1 g yeast extract (Difco), 2 g tryptone (Difco) and 15 g agar (Difco) per liter distilled water. Spores of each strain obtained from slant cultures on BA medium were lyophilized and used as inocula for mycelial growth. Mycelia for protoplast formation were obtained by growing Streptoverticillium strains in TS broth containing 30 g Bacto Tryptic Soy Broth (Difco) per liter distilled water, on a rotary shaker (200 rpm) at 28°C. Mycelial growth was estimated at 3 h intervals by determining OD580. The strains were cultivated in TS broth in the absence of glycine or supplemented with 0.75 or 1.5% (w/v) of this aminoacid. The mycelia were harvested by centrifugation and washed three times with 0.5 M sucrose (Sigma). 2.2. Effect of different conditions on protoplast formation and regeneration Protoplast preparation was achieved by washing mycelia (1 g wet weight) from different steps of the growth phase. The pellets were incubated at 28°C for 2 h in 8 ml of Sagara protoplast medium [7] ('P' medium; 0.01 M phosphate buffer (pH 7.0), 0.1 M MgSO4, 1.0 M sucrose) with the addition of different concentrations (5, 10 and 20 mg/ml) of lysozyme (Sigma). Different concentrations of divalent cations Ca 2+ (25 mM CaC12), Mg z+ (10 mM MgSO4.7H20), Mn 2+ (10 mM MnSO4.H20) and Zn 2+ (10 mM Zn SO4.7H20) and osmotic stabilizers (0.25, 0.5 and 1.0 M sucrose, Sigma), 0.5 M inositol (Sigma) or 0.5 M mannitol (Sigma)) were assayed by modifying the original P medium and incubating mycelia for 1 h. Protoplast formation and estimation were monitored by phase-contrast microscopy in a Thoma counter chamber. After determining protoplast concentration, the suspension was centrifuged at 3000 × g for 5 min at 4°C. The pellets were washed twice with P medium to eliminate the lysozyme.
A.-K. Scheu et al./Journal of MicrobiologicalMethods 19 (1994) 197-206
199
The viability of protoplasts (2 x 108 protoplasts/ml) from each strain was estimated by diluting them in 2 ml of P medium or 0.01% SDS (Sigma) (w/v) in water. Protoplasts diluted in P medium were plated on BA medium supplemented with 0.5 M sucrose (BAS medium), while those diluted in distilled water/SDS were plated on BA medium without any osmotic stabilizer. Thus, contamination of the protoplasts with fragments of mycelium could be detected and regeneration expressed as the percentage of colony forming protoplasts out of the total number of protoplasts plated (3 × 103 per plate). Plates were incubated at 28°C for 36 h. Protoplast regeneration was carried out on the classic regeneration media R1, R2, R3 and SS, described for Streptomyces [8-10] and on BAS medium. 2.3. Optimal conditions for protoplast regeneration In order to select the optimal conditions for protoplast regeneration, the effect of different modifications of the original BA medium were studied. These changes were as follows: agar overlay techniques and partial dehydration; presence of 0.5 M sucrose or 0.5 M disodium succinate (Sigma) as stabilizers and Mg 2+ (40 mM MgC12.6H20) and/or Ca 2+ (15 mM CaC12.2HzO), at the same concentrations as in the classic regeneration media RI, R2, R3 and SS for Streptomyces. The effect of these factors is expressed as the percentage of regeneration achieved on BAS medium. For the overlay assay, Petri plates containing undehydrated and partially dehydrated BAS medium were overlayered with 8 ml of the same medium with a lower agar concentration (4 mg/ml). Dehydration effect was studied by incubating BAS medium plates at 50°C to reach 20% loss of weight.
N C I M B 40447
CECT 3263
12 Abs, 1580 nm~
'il C~
~
.
CECT 3265 10 Abs. ~580 nm~
10 "I~L l'~l~ Nm)
6-
6
4-
4 2
.
.
.
.
70
~=
10
20
1"~e (bourn)
!
40 40 Time 5'~um)
50
40
70
c"d"
1o~
~
=
,~
so
40
70
Tame (hour,s)
Control
.e-Glycine 0.5% -A-Glycine 1% -a-Glycine 1.5%
Fig. 1. Mycelial time course of the three Streptoverticillium strains in TS broth and in the presence of different concentrations of glycine. The cultures were diluted with fresh medium in order to reach an adequated absorbance value to measure in the spectrophotometer.
200
A.-K. Scheu et al./Journal of Microbiological
Methodv 19 (1994) 197-206
A.-K. Scheu et al./Journal of MicrobiologicalMethods 19 (1994) 197-206
201
Table 1 Regeneration frequencies (%) of protoplasts from Streptoverticilliumstrains NCIMB 40447, CECT 3263 and CECT 3265 on the classic regeneration media (R1, R2, R3, and SS) and on BAS medium Regeneration media RI
R2
R3
SS
BAS
NCIMB 40447
0
0
0.08
0.15
0.17
CECT 3263
0
0
0
0.46
1.02
CECT 3265
0
0
0
0.41
1.18
Regeneration frequencies (%) are expressedas the percentage of colony forming protoplasts observed out of the total number of protoplasts plated. All results are means of triplicate values. 3. R e s u l t s
3.1. Glycine effect on cell growth The time course of mycelial growth for the three Streptoverticillium strains in TS broth (control) and in the presence of different concentrations of glycine is represented in Fig. 1. 3.2. Protoplast formation and regeneration in different conditions Protoplasts were formed from each of the three Streptoverticillium strains following incubation with lysozyme in an hypertonic stabilizing solution. Conversion of mycelia to protoplasts was completed in about 1 h. Fig. 2 shows sequential protoplast formation from S. griseocarneum mycelium. Since protoplast formation from mycelia at a later or earlier growth phase than the mid-exponential one decreased greatly, the subsequent assays were performed with mycelia taken from the midexponential phase of growth. Only mycelia from this growth phase furnished viable protoplasts, while those from mycelia at other growth phases were negligible. The optimum lysozyme concentration for each strain was 10 mg/ml. Smaller or larger amounts of lysozyme decreased the protoplast yields. The viability of the protoplasts produced in P medium supplemented with 10 mg/ml lysozyme depended on incubation time and strain. M a x i m u m regeneration values were achieved in strains 40447, 3263 and 3265 after 90, 60 and 120 min of incubation with lysozyme, respectively (data not shown). Protoplasts did not grow into colonies following inoculation onto the classic R1 and R2 media, generally used for regenerating Streptomyces protoplasts, and in the R3 and SS media regeneration, although growth was present, was very p o o r (Table 1). The earliest success in obtaining regeneration of Streptoverticillium protoplasts
4--
Fig. 2. Myceliumphase-contrast micrographs of NCIMB 40447 strain incubated in the absence (A) or in presence of lysozymefor 5 (B), 30 (C) or 60 (D) min.
A.-K. Scheu et al./Journal of Microbiological Methods 19 (1994) 197-206
202
B
A Protoplasts/ml(xlO')
Regenerationfrequency (%)
70 ¸
, 100
60.
8O 50-
60
40-
30-
40
20-
20 10-
0
NCIMB 40447
CECT3263
CECT3265
0
'"'"=
NCIMB 40447
CECT3263
CECT3265
rrmAbsence of glycine 1--10.75% glycine E31.5% glycine Fig. 3. Protoplast formation (A) and regeneration (B) in the three Streptoverticillium strains. Mycelia were previously grown in TS broth with different concentrations of glycine.
was obtained by spreading cells onto the surface of BAS medium. Colonies from protoplasts became visible after 30 h of incubation at 28°C. Protoplast formation achieved maximum values when mycelia were cultured in the presence of 1.5% (w/v) glycine (Fig. 3). However, protoplasts produced from mycelia grown in glycine supplemented TS broth did not regenerate in any of the strains studied. Protoplasts from the three strains could be stabilized during lysozyme digestion with sucrose, inositol or mannitol. However, only protoplasts stabilized with sucrose produced significant regeneration values (Fig. 4). Although protoplast formation is possible in the absence of Ca + z, these protoplasts are not able to regenerate viable cells. Strains 40447 and 3265 required Mg +2 during protoplast formation, while strain 3263 did not. However, strain 3263 protoplasts produced in the absence of Mg +2 regenerated in a lower number than those produced in the presence of this divalent ion. The presence of Zn z ÷ or Mn ÷ 2 during lysozyme digestion did not permit protoplast formation in any of the strains tested.
3.3. Optimal conditions for regeneration on BA medium Results of the effect of different culture conditions (osmotic stabilizers, divalent cations, partial dehydration and agar overlay techniques) on the regeneration of Streptoverticillium protoplasts are shown in Table 2.
A.-K, Scheu et al./Journal of Microbiological Methods 19 (1994) 197-206
203
B
A Protoplasts/ml (xlO')
100
Regenerationfrequency (%)
80
80
30 40
20-
20 10-
iiiiii]1
NClMB 40447
CECT3263
CECT3265
NClMB 40447
CECT3268
CECT 3265
rr~0.5 M sucrose IZ]0.5 M inositol
E30.5 M mannitol Fig. 4. Effect of different osmotic stabilizers on formation (A) and regeneration (B) of Streptoverticillium protoplasts produced in P medium.
In none of the three strains could protoplast regeneration be detected in BA medium stabilized with disodium succinate. Regeneration increased greatly in strains 40447 and 3265, when protoplasts were plated on an overlayer of BAS medium containing 0.4%(w/v) agar. Partially dehydrated BAS medium (20%)did not permit Streptoverticillium protoplasts to regenerate. Table 2 Effect of differentconditions on protoplast regeneration in the three strains of Streptoverticillium using BAS medium as 100% baseline Strain
BAS (control) BA + succinate Dehydrated BAS Overlayed BAS BAS + Ca +2 BAS + Mg ÷2 BAS + Ca ÷2 + M g ÷2
N C I M B 40447
C E C T 3263
C E C T 3265
100 0 0 199 120 146 65
100 0 0 57 27 33 24
100 0 48 124 106 51 69
204
A.-K. Scheu et al./Journal of Microbiological Methods 19 (1994) 197-206
The presence of Ca 2+ in the regeneration medium improved regeneration yields slightly in strains 40447 (120%) and 3265 (106%), while strain 3263 protoplasts regenerated very poorly. The presence of Mg 2+ improved the regeneration values in strain 40447 (146%). Moreover, colonies from regenerated protoplasts in Mg 2÷ supplemented BAS medium appeared to be much smaller than in the absence of this cation. The addition of both cations, Mg 2÷ and Ca 2÷, to the regeneration medium, seemed to have an antagonistic effect on the protoplast regeneration in strains 40447 (65%) and 3265 (70%). However, although the presence of both cations resulted in a poor regeneration in strain 3263, it is higher than if only one was present. The presence of both cations resulted in a poor regeneration in the strain 3263, similar to that resulted with the addition of only one ion.
4. Discussion
This report describes a procedure for protoplast production and regeneration in Streptoverticillium strains. In general, regeneration media described for Streptomyces protoplasts were found to be unsuccessful. In SS medium, containing complex nitrogen and carbon sources and sucrose as stabilizer, poor regeneration was detected. BAS medium appeared to furnish optimal environmental and nutritional requirements for protoplast regeneration in Streptoverticillium. These results suggest that complex nitrogen or carbon sources are required for efficient cell regeneration in this genus. Further studies will elucidate the nutritional requirements for protoplast regeneration in Streptoverticillium. Sagara [7] reported that the presence of glycine in the growth medium increases the mycelia sensitivity to lysozyme and the published procedures for preparing mycelial cultures for protoplast formation usually involve mycelial growth in media containing glycine [1,9,10]. In contrast, the presence of glycine during mycelial growth enhances protoplast formation in Streptoverticillium, but it does not furnish viable protoplasts. The high sensitivity of Streptoverticillium to the presence of glycine during cell growth indicates the replacement of D-alanine residues by glycine in the peptidoglycan, thereby interfering with cross-linking in the cell wall [11]. Previous workers have commented on the need to allow the protoplasts to retain the hypothetical primer or residual cell wall material, if growth of the cell wall (regeneration) was to be achieved [12]. The results of our experiments may be attributed to the total removal of this residual cell wall material, when mycelium is grown in a medium containing glycine. It was necessary to treat Streptoverticillium mycelia with much higher concentrations of lysozyme than described for most of the Streptomyces strains [1,10,13] to achieve cell wall degeneration and protoplast formation. Differences in lysozyme sensitivity between members of the two genera have been reported, but they cannot be explained in terms of different cell wall acyl types [14]. Examination of the efficiency of cell regeneration in Streptoverticillium protoplasts throughout the growth cycle, suggested that a physiological state for optimum cell regeneration is reached during the exponential phase of growth, while
A.-K. Scheu et al./Journal of Microbiological Methods 19 (1994) 197-206
205
protoplasts from later stages only regenerate very poorly. However, maximal regeneration rates for the Streptomyces protoplasts are achieved when the mycelium is grown until the transition stage between the exponential and the stationary phases of growth [15]. It could be considered that the accumulation of secondary metabolites like polyenes and pigments on the peptidoglycan may reduce the target points for lysozyme activity in Streptoverticillium [4,16]. Although the stabilizer for optimal protoplast production in Streptoverticillium depends on the strain, the yield of regenerated protoplasts was greater when sucrose rather than inositol or mannitol was used as stabilizer. Previous reports on Streptomyces state that the stabilizer for optimal protoplast formation and cell regeneration can be strain specific [13]. Magnesium is required for protoplast formation and regeneration in Streptoverticillium strains. The effect of this ion on the stabilization of protoplasts might be related to the prevention of lipid residue release from the plasmatic membrane [10]. Although protoplast production is possible in the absence of calcium, it can be concluded that the combination of both ions is effective in preparing and stabilizing Streptoverticillium protoplasts in hypertonic solutions. Further studies carried out by modifying the stabilized BAS regeneration medium show differences in the optimal regeneration conditions between the genera Streptomyces and Streptoverticillium. The regeneration of Streptomyces protoplasts has been described to improve greatly in media stabilized with succinate and using thin agar overlay techniques [9]. While the use of a thin agar overlayer of BAS medium was found to enhance regeneration greatly in strains 40447 and 3265, the presence of succinate as stabilizer does not allow Streptoverticillium protoplasts to regenerate. The use of partial dehydration conditions and agar overlayer techniques, increases greatly the regeneration frequencies in Streptomyces, which can be explained in terms of a lower diffusion of auto-inhibitory substances [15]. Since protoplasts of Streptoverticillium can even develop into confluent lawns, no auto-inhibition was found. However, protoplasts did not regenerate in a partially dehydrated medium. This suggests that none of the tested nutritional sources required for cell regeneration of Streptoverticillium protoplasts diffused or allowed regeneration. The effect of magnesium and/or calcium ions on the protoplast regeneration is strain dependent. Meaningfully, colonies from regenerated protoplasts in BAS medium supplemented with magnesium appeared to be much smaller than in the absence of this ion. This effect can not be explained in terms of the usual mechanism of action described by this ion.
Acknowledgements The authors are indebted to Prof. F. Uruburu for kindly providing the Streptoverticillium strains from the Spanish Culture Type Collection (CECT) and to C.F. Warren (ICE, Alcal~i de Henares) for the linguistic assistance.
206
A.-K. Scheu et al./Journal of Microbiological Methods 19 (1994) 197-206
References [1] Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Bruton, C.J., Kieser, H.M., Lydiate, D.J., Smith, C.P., Ward, J.M. and Schrempf, H. (1985) Genetic manipulation of Streptomyces, pp. 1-33. A Laboratory Manual. John Innes Foundation, Norwich. [2] Locci, R., and Schofield G.M. (1989) Genus Streptoverticillium. In: Bergey's Manual of Systematic Bacteriology, Volume 4 (Williams, S.T., Sharpe, M.E. and Holt, J.G.), pp. 2492-2504, Williams & Wilkins, Baltimore. [3] Locci, R., Baldacci, E. and Petrolini-Baldan, B. (1969) The genus Streptoverticillium. A taxonomic study. J. Microbiol. 17, 1-60. [4] Soliveri, J., Arias, M.E. and Laborda, F. (1987) PA-5 and PA-7, pentaene and heptaene macrolide antibiotics produced by a new isolate of Streptoverticillium from Spanish soil. Appl. Microbiol. Biotechnol. 25, 366-371. [5] Fidalgo, M.L., Alonso, J.L., Soliveri, J. and Arias, M.E. (1992) APHE-1 and APHE-2, two new antimicrobial and cytocidal antibiotics. I. Taxonomy, fermentation, isolation and biological activity. J. Antibiotics 45, 1753-1758. [6] Fidalgo, M.L., Arias, M.S., Soliveri, J. and Arias, M.E. (1992) APHE-I and APHE-2, two new antimicrobial and cytocidal antibiotics. II. Physicochemical properties and structure elucidation. J. Antibiotics. 45, 1759-1762. [7] Sagara, Y., Fukui, K., Ota, F., Yoshida, N., Kashiyama, T. and Fujimoto, M. (1971) Rapid formation of protoplasts of Streptomyces griseoflavus and their fine structure. Japan. J. Microbiol. 15, 73-84. [8] Schrempf, H. (1982) Plasmid loss and changes within the chromosomal DNA of Streptomyces reticuli. J. Bacteriol. 151,701-707. [9] Shirahama, T., Furumai, T. and Okanishi, M. (1981) A modified regeneration method for streptomycete protoplasts. Agric. Biol. Chem. 45, 1271-1273. [10] Okanishi, M., Suzuki, K. and Umezawa, H. (1974) Formation and reversion of streptomycete protoplasts: cultural conditions and morphological study. J. Gen. Microbiol. 80, 389-400. [11] Hammes, W., Schleifer, K.H. and Kandler, O. (1973) Mode of action of glycine on the biosynthesis of peptidoglycan. J. Bacteriol. 116, 1029-1053. [12] Lee-Wickner, L. and Chassy, B.M. (1984) Production and regeneration of Lactobacillus casei protoplasts. Appl. Environ. Microbiol. 48, 994-1000. [13] Hooley, P. and Wellington, A.M.H. (1985) Formation and regeneration of protoplasts of Streptomyces hygroscopicus. Lett. Appl. Microbiol. 1, 77-80. [14] Surrey, A., Attwell, R.W. and Cross, T. (1985) Cell wall acyl type in some strains of Streptoverticillium and Streptomyces. J. Gen. Appl. Microbiol. 31,493-497. [15] Baltz, R.H. and Matshushima,P.(1981) Protoplast fusion in Streptomyces: conditions for efficient genetic recombination and cell regeneration. J. Gen. Microbiol. 127, 137-146. [16] Cherny, N.E., Tikkonenko, A.S. and Kalakoutskii, L.V. (1984) Comparative structural characterization of material extractable from aerial sheath of Streptomyces roseoflavus var. roseofungini. Arch. Microbiol. 101, 71-82.
Journal of Microbiological Methods 19 (1994) 197-206
Factors affecting protoplast formation and regeneration in three strains of Streptoverticillium A n n - K a r o l i n Scheu, J u a n Soliveri *, M a r i a - E n r i q u e t a A r i a s Departamento de Microbiologia y Parasitologia. Universidadde Alcal6 de Henares, 28871-Alcal~ de Henares, Madrid, Spain
Received 25 May 1993; revision received 2 October 1993; accepted 5 October 1993
Abstract
Conditions are reported for efficient protoplast formation and regeneration in three strains of Streptoverticillium. Mid-exponential phase mycelia, grown in Tryptic-soy broth supplemented with 1.5% (w/v) glycine, were highly sensitive to lysozyme, but the protoplasts did not regenerate. The presence of Ca ÷2 in protoplast formation media was required for cell regeneration. Mg +2 improved the regeneration yield only in the case of Streptoverticillium griseocarneum NCIMB 40447. Protoplasts only regenerated in media containing complex nitrogen and carbon sources. Maximum cell regeneration was achieved with protoplasts obtained from mid-exponential growth phase mycelia and plated on an undehydrated Bennet agar overlayered with the same medium containing a lower agar concentration. Sucrose proved to be the optimal osmotic stabilizer and was added to the media for protoplast formation and regeneration. Key words: Streptoverticillium; Protoplast production; Protoplast regeneration
I. Introduction
One of the basic techniques for D N A cloning in actinomycetes is protoplast preparation for the efficient regeneration of normal mycelia to carry out protoplast transformation or fusion and so bypass the barriers encountered with other methods. The development of these techniques for Streptomyces species [1] has considerably enhanced the potential for the genetic manipulation of this industrially important genus. Although Streptoverticillium strains can produce several antibiotics [2], ~ponding
author. Tel: 34.1.8854693. Fax: 34.1.8854660. Email: [email protected]
0167-7012/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 7 0 1 2 ( 9 3 ) E 0 0 6 5 - Z
198
A.-K. Scheuet al./Journalof MicrobiologicalMethods 19 (1994) 197-206
no investigations have addressed protoplast formation. In this sense, and in order to compare the production and regeneration of protoplasts in both genera, classical procedures described for Streptomyces have been tested in three species of Streptoverticillium. The two very closely related genera, Streptomyces and Streptoverticillium, show differences in morphology and physiology [2,3]. In this report, efficient reliable methods for protoplast formation and regeneration in Streptoverticillium strains, based on techniques described for Streptomyces, have been developed. Some differences among factors affecting these processes with either genera have been also established.
2. Materials and methods
2.1. Bacterial strains, mycelial growth and glycine effect In this study three antibiotic producing strains were used: Streptoverticillium griseocarneum NCIMB 40447, an isolate from Spanish soil [4,5,6], Streptoverticillium ladakanum CECT 3263 and Streptoverticillium netropsis CECT 3265. These strains share a high phenotypic similarity and will be referred to hereafter as strain 40447, 3263 and 3265, respectively. The strains were stock-cultured on a modified Bennet agar (BA) medium [3] which contained 10 g glucose (Merck), 1 g Lab-Lenko powder (Oxoid), 1 g yeast extract (Difco), 2 g tryptone (Difco) and 15 g agar (Difco) per liter distilled water. Spores of each strain obtained from slant cultures on BA medium were lyophilized and used as inocula for mycelial growth. Mycelia for protoplast formation were obtained by growing Streptoverticillium strains in TS broth containing 30 g Bacto Tryptic Soy Broth (Difco) per liter distilled water, on a rotary shaker (200 rpm) at 28°C. Mycelial growth was estimated at 3 h intervals by determining OD580. The strains were cultivated in TS broth in the absence of glycine or supplemented with 0.75 or 1.5% (w/v) of this aminoacid. The mycelia were harvested by centrifugation and washed three times with 0.5 M sucrose (Sigma). 2.2. Effect of different conditions on protoplast formation and regeneration Protoplast preparation was achieved by washing mycelia (1 g wet weight) from different steps of the growth phase. The pellets were incubated at 28°C for 2 h in 8 ml of Sagara protoplast medium [7] ('P' medium; 0.01 M phosphate buffer (pH 7.0), 0.1 M MgSO4, 1.0 M sucrose) with the addition of different concentrations (5, 10 and 20 mg/ml) of lysozyme (Sigma). Different concentrations of divalent cations Ca 2+ (25 mM CaC12), Mg z+ (10 mM MgSO4.7H20), Mn 2+ (10 mM MnSO4.H20) and Zn 2+ (10 mM Zn SO4.7H20) and osmotic stabilizers (0.25, 0.5 and 1.0 M sucrose, Sigma), 0.5 M inositol (Sigma) or 0.5 M mannitol (Sigma)) were assayed by modifying the original P medium and incubating mycelia for 1 h. Protoplast formation and estimation were monitored by phase-contrast microscopy in a Thoma counter chamber. After determining protoplast concentration, the suspension was centrifuged at 3000 × g for 5 min at 4°C. The pellets were washed twice with P medium to eliminate the lysozyme.
A.-K. Scheu et al./Journal of MicrobiologicalMethods 19 (1994) 197-206
199
The viability of protoplasts (2 x 108 protoplasts/ml) from each strain was estimated by diluting them in 2 ml of P medium or 0.01% SDS (Sigma) (w/v) in water. Protoplasts diluted in P medium were plated on BA medium supplemented with 0.5 M sucrose (BAS medium), while those diluted in distilled water/SDS were plated on BA medium without any osmotic stabilizer. Thus, contamination of the protoplasts with fragments of mycelium could be detected and regeneration expressed as the percentage of colony forming protoplasts out of the total number of protoplasts plated (3 × 103 per plate). Plates were incubated at 28°C for 36 h. Protoplast regeneration was carried out on the classic regeneration media R1, R2, R3 and SS, described for Streptomyces [8-10] and on BAS medium. 2.3. Optimal conditions for protoplast regeneration In order to select the optimal conditions for protoplast regeneration, the effect of different modifications of the original BA medium were studied. These changes were as follows: agar overlay techniques and partial dehydration; presence of 0.5 M sucrose or 0.5 M disodium succinate (Sigma) as stabilizers and Mg 2+ (40 mM MgC12.6H20) and/or Ca 2+ (15 mM CaC12.2HzO), at the same concentrations as in the classic regeneration media RI, R2, R3 and SS for Streptomyces. The effect of these factors is expressed as the percentage of regeneration achieved on BAS medium. For the overlay assay, Petri plates containing undehydrated and partially dehydrated BAS medium were overlayered with 8 ml of the same medium with a lower agar concentration (4 mg/ml). Dehydration effect was studied by incubating BAS medium plates at 50°C to reach 20% loss of weight.
N C I M B 40447
CECT 3263
12 Abs, 1580 nm~
'il C~
~
.
CECT 3265 10 Abs. ~580 nm~
10 "I~L l'~l~ Nm)
6-
6
4-
4 2
.
.
.
.
70
~=
10
20
1"~e (bourn)
!
40 40 Time 5'~um)
50
40
70
c"d"
1o~
~
=
,~
so
40
70
Tame (hour,s)
Control
.e-Glycine 0.5% -A-Glycine 1% -a-Glycine 1.5%
Fig. 1. Mycelial time course of the three Streptoverticillium strains in TS broth and in the presence of different concentrations of glycine. The cultures were diluted with fresh medium in order to reach an adequated absorbance value to measure in the spectrophotometer.
200
A.-K. Scheu et al./Journal of Microbiological
Methodv 19 (1994) 197-206
A.-K. Scheu et al./Journal of MicrobiologicalMethods 19 (1994) 197-206
201
Table 1 Regeneration frequencies (%) of protoplasts from Streptoverticilliumstrains NCIMB 40447, CECT 3263 and CECT 3265 on the classic regeneration media (R1, R2, R3, and SS) and on BAS medium Regeneration media RI
R2
R3
SS
BAS
NCIMB 40447
0
0
0.08
0.15
0.17
CECT 3263
0
0
0
0.46
1.02
CECT 3265
0
0
0
0.41
1.18
Regeneration frequencies (%) are expressedas the percentage of colony forming protoplasts observed out of the total number of protoplasts plated. All results are means of triplicate values. 3. R e s u l t s
3.1. Glycine effect on cell growth The time course of mycelial growth for the three Streptoverticillium strains in TS broth (control) and in the presence of different concentrations of glycine is represented in Fig. 1. 3.2. Protoplast formation and regeneration in different conditions Protoplasts were formed from each of the three Streptoverticillium strains following incubation with lysozyme in an hypertonic stabilizing solution. Conversion of mycelia to protoplasts was completed in about 1 h. Fig. 2 shows sequential protoplast formation from S. griseocarneum mycelium. Since protoplast formation from mycelia at a later or earlier growth phase than the mid-exponential one decreased greatly, the subsequent assays were performed with mycelia taken from the midexponential phase of growth. Only mycelia from this growth phase furnished viable protoplasts, while those from mycelia at other growth phases were negligible. The optimum lysozyme concentration for each strain was 10 mg/ml. Smaller or larger amounts of lysozyme decreased the protoplast yields. The viability of the protoplasts produced in P medium supplemented with 10 mg/ml lysozyme depended on incubation time and strain. M a x i m u m regeneration values were achieved in strains 40447, 3263 and 3265 after 90, 60 and 120 min of incubation with lysozyme, respectively (data not shown). Protoplasts did not grow into colonies following inoculation onto the classic R1 and R2 media, generally used for regenerating Streptomyces protoplasts, and in the R3 and SS media regeneration, although growth was present, was very p o o r (Table 1). The earliest success in obtaining regeneration of Streptoverticillium protoplasts
4--
Fig. 2. Myceliumphase-contrast micrographs of NCIMB 40447 strain incubated in the absence (A) or in presence of lysozymefor 5 (B), 30 (C) or 60 (D) min.
A.-K. Scheu et al./Journal of Microbiological Methods 19 (1994) 197-206
202
B
A Protoplasts/ml(xlO')
Regenerationfrequency (%)
70 ¸
, 100
60.
8O 50-
60
40-
30-
40
20-
20 10-
0
NCIMB 40447
CECT3263
CECT3265
0
'"'"=
NCIMB 40447
CECT3263
CECT3265
rrmAbsence of glycine 1--10.75% glycine E31.5% glycine Fig. 3. Protoplast formation (A) and regeneration (B) in the three Streptoverticillium strains. Mycelia were previously grown in TS broth with different concentrations of glycine.
was obtained by spreading cells onto the surface of BAS medium. Colonies from protoplasts became visible after 30 h of incubation at 28°C. Protoplast formation achieved maximum values when mycelia were cultured in the presence of 1.5% (w/v) glycine (Fig. 3). However, protoplasts produced from mycelia grown in glycine supplemented TS broth did not regenerate in any of the strains studied. Protoplasts from the three strains could be stabilized during lysozyme digestion with sucrose, inositol or mannitol. However, only protoplasts stabilized with sucrose produced significant regeneration values (Fig. 4). Although protoplast formation is possible in the absence of Ca + z, these protoplasts are not able to regenerate viable cells. Strains 40447 and 3265 required Mg +2 during protoplast formation, while strain 3263 did not. However, strain 3263 protoplasts produced in the absence of Mg +2 regenerated in a lower number than those produced in the presence of this divalent ion. The presence of Zn z ÷ or Mn ÷ 2 during lysozyme digestion did not permit protoplast formation in any of the strains tested.
3.3. Optimal conditions for regeneration on BA medium Results of the effect of different culture conditions (osmotic stabilizers, divalent cations, partial dehydration and agar overlay techniques) on the regeneration of Streptoverticillium protoplasts are shown in Table 2.
A.-K, Scheu et al./Journal of Microbiological Methods 19 (1994) 197-206
203
B
A Protoplasts/ml (xlO')
100
Regenerationfrequency (%)
80
80
30 40
20-
20 10-
iiiiii]1
NClMB 40447
CECT3263
CECT3265
NClMB 40447
CECT3268
CECT 3265
rr~0.5 M sucrose IZ]0.5 M inositol
E30.5 M mannitol Fig. 4. Effect of different osmotic stabilizers on formation (A) and regeneration (B) of Streptoverticillium protoplasts produced in P medium.
In none of the three strains could protoplast regeneration be detected in BA medium stabilized with disodium succinate. Regeneration increased greatly in strains 40447 and 3265, when protoplasts were plated on an overlayer of BAS medium containing 0.4%(w/v) agar. Partially dehydrated BAS medium (20%)did not permit Streptoverticillium protoplasts to regenerate. Table 2 Effect of differentconditions on protoplast regeneration in the three strains of Streptoverticillium using BAS medium as 100% baseline Strain
BAS (control) BA + succinate Dehydrated BAS Overlayed BAS BAS + Ca +2 BAS + Mg ÷2 BAS + Ca ÷2 + M g ÷2
N C I M B 40447
C E C T 3263
C E C T 3265
100 0 0 199 120 146 65
100 0 0 57 27 33 24
100 0 48 124 106 51 69
204
A.-K. Scheu et al./Journal of Microbiological Methods 19 (1994) 197-206
The presence of Ca 2+ in the regeneration medium improved regeneration yields slightly in strains 40447 (120%) and 3265 (106%), while strain 3263 protoplasts regenerated very poorly. The presence of Mg 2+ improved the regeneration values in strain 40447 (146%). Moreover, colonies from regenerated protoplasts in Mg 2÷ supplemented BAS medium appeared to be much smaller than in the absence of this cation. The addition of both cations, Mg 2÷ and Ca 2÷, to the regeneration medium, seemed to have an antagonistic effect on the protoplast regeneration in strains 40447 (65%) and 3265 (70%). However, although the presence of both cations resulted in a poor regeneration in strain 3263, it is higher than if only one was present. The presence of both cations resulted in a poor regeneration in the strain 3263, similar to that resulted with the addition of only one ion.
4. Discussion
This report describes a procedure for protoplast production and regeneration in Streptoverticillium strains. In general, regeneration media described for Streptomyces protoplasts were found to be unsuccessful. In SS medium, containing complex nitrogen and carbon sources and sucrose as stabilizer, poor regeneration was detected. BAS medium appeared to furnish optimal environmental and nutritional requirements for protoplast regeneration in Streptoverticillium. These results suggest that complex nitrogen or carbon sources are required for efficient cell regeneration in this genus. Further studies will elucidate the nutritional requirements for protoplast regeneration in Streptoverticillium. Sagara [7] reported that the presence of glycine in the growth medium increases the mycelia sensitivity to lysozyme and the published procedures for preparing mycelial cultures for protoplast formation usually involve mycelial growth in media containing glycine [1,9,10]. In contrast, the presence of glycine during mycelial growth enhances protoplast formation in Streptoverticillium, but it does not furnish viable protoplasts. The high sensitivity of Streptoverticillium to the presence of glycine during cell growth indicates the replacement of D-alanine residues by glycine in the peptidoglycan, thereby interfering with cross-linking in the cell wall [11]. Previous workers have commented on the need to allow the protoplasts to retain the hypothetical primer or residual cell wall material, if growth of the cell wall (regeneration) was to be achieved [12]. The results of our experiments may be attributed to the total removal of this residual cell wall material, when mycelium is grown in a medium containing glycine. It was necessary to treat Streptoverticillium mycelia with much higher concentrations of lysozyme than described for most of the Streptomyces strains [1,10,13] to achieve cell wall degeneration and protoplast formation. Differences in lysozyme sensitivity between members of the two genera have been reported, but they cannot be explained in terms of different cell wall acyl types [14]. Examination of the efficiency of cell regeneration in Streptoverticillium protoplasts throughout the growth cycle, suggested that a physiological state for optimum cell regeneration is reached during the exponential phase of growth, while
A.-K. Scheu et al./Journal of Microbiological Methods 19 (1994) 197-206
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protoplasts from later stages only regenerate very poorly. However, maximal regeneration rates for the Streptomyces protoplasts are achieved when the mycelium is grown until the transition stage between the exponential and the stationary phases of growth [15]. It could be considered that the accumulation of secondary metabolites like polyenes and pigments on the peptidoglycan may reduce the target points for lysozyme activity in Streptoverticillium [4,16]. Although the stabilizer for optimal protoplast production in Streptoverticillium depends on the strain, the yield of regenerated protoplasts was greater when sucrose rather than inositol or mannitol was used as stabilizer. Previous reports on Streptomyces state that the stabilizer for optimal protoplast formation and cell regeneration can be strain specific [13]. Magnesium is required for protoplast formation and regeneration in Streptoverticillium strains. The effect of this ion on the stabilization of protoplasts might be related to the prevention of lipid residue release from the plasmatic membrane [10]. Although protoplast production is possible in the absence of calcium, it can be concluded that the combination of both ions is effective in preparing and stabilizing Streptoverticillium protoplasts in hypertonic solutions. Further studies carried out by modifying the stabilized BAS regeneration medium show differences in the optimal regeneration conditions between the genera Streptomyces and Streptoverticillium. The regeneration of Streptomyces protoplasts has been described to improve greatly in media stabilized with succinate and using thin agar overlay techniques [9]. While the use of a thin agar overlayer of BAS medium was found to enhance regeneration greatly in strains 40447 and 3265, the presence of succinate as stabilizer does not allow Streptoverticillium protoplasts to regenerate. The use of partial dehydration conditions and agar overlayer techniques, increases greatly the regeneration frequencies in Streptomyces, which can be explained in terms of a lower diffusion of auto-inhibitory substances [15]. Since protoplasts of Streptoverticillium can even develop into confluent lawns, no auto-inhibition was found. However, protoplasts did not regenerate in a partially dehydrated medium. This suggests that none of the tested nutritional sources required for cell regeneration of Streptoverticillium protoplasts diffused or allowed regeneration. The effect of magnesium and/or calcium ions on the protoplast regeneration is strain dependent. Meaningfully, colonies from regenerated protoplasts in BAS medium supplemented with magnesium appeared to be much smaller than in the absence of this ion. This effect can not be explained in terms of the usual mechanism of action described by this ion.
Acknowledgements The authors are indebted to Prof. F. Uruburu for kindly providing the Streptoverticillium strains from the Spanish Culture Type Collection (CECT) and to C.F. Warren (ICE, Alcal~i de Henares) for the linguistic assistance.
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