Effect of medium composition on the maintenance of a recombinant plasmid in Bacillus subtilis Yuval Shoham and Arnold L. Demain Fermentation Microbiology Laboratory, D e p a r t m e n t o f Biology, M a s s a c h u s e t t s Institute o f Technology, Cambridge, M A
Recombinant plasmid pCED3 [confers ~-galactosidase production (LacZ +) and kanamycin resistance (Km")] in Bacillus subtilis was found to be both segregationally and structurally unstable. Since many solutions to segregational instability are already available, the problem of structural instability was specifically addressed by inclusion of kanamycin in the growth media. Culture instability was found to be highest in complex and defined media supporting high growth rates. Stabilization over the duration of the experiment (40 generations) was achieved by use of a recently developed chemically defined medium supporting a lower growth rate. Slowing down growth by decreasing temperature was much less effective. A major effect of the growth medium appears to be that of decreasing the growth rate advantage held by cells with plasmid deletions over parental cells containing the intact plasmid.
Keywords: Recombinantplasmid; Bacillus subtilis; plasmid instability
Introduction Three aspects of Bacillus are of special interest for potential industrial applications: (1) Bacillus subtilis has no known pathogenic interactions with humans or animals. 1The only authentic report of human infection due toB. subtilis was in a severely compromised host, a drug addict. 2 Unlike Gram-negative organisms, the cell surface of B. subtilis is composed only of peptidoglycan and teichoic acid.3 Therefore, recombinant DNA products produced by B. subtilis would not be contaminated by lipopolysaccharide endotoxins. 4 (2)B. subtilis is able to secrete homologous and heterologous proteins .5-9 (3) Bacillus strains have many industrial applications, and systems are well developed for large-scale cultivation. ~0Their potential in commercial genetic engineering has been reviewed by Workman et al. ~ The instability of cultures containing recombinant
The present address of Dr. Shoham is the Department of Food Engineering and Biotechnology,The Technion, Haifa 32000, Israel Address reprintrequests to Dr. Demainat the FermentationMicrobiology Laboratory Department of Biology, Massachusetts Institute of Technology,Cambridge, MA 02139 Received 5 January 1989; revised 30 May 1989 330
Enzyme Microb. Technol., 1990, vol. 12, May
plasmids is a problem often encountered in applying recombinant DNA technology.~2 During growth or storage, a population of plasmid-bearing cells often segregates to give cells that are either plasmid-free or contain modified plasmids. During large-scale production of recombinant DNA products, plasmid instability directly decreases the efficiency and productivity of the fermentation process. 13,14Industrial fermentations typically involve a large number of generations (over 50), allowing ample time for modified cells to take over the population. Especially during the manufacturing of pharmaceuticals which require GMP (Good Manufacturing Practice) conditions, the drug regulatory authorities consider plasmid instability to be undesirable. 15 Two general mechanisms are responsible for plasmid instability. First, plasmids may be lost from some cells due to unequal segregation (i.e. segregational instability). Second, plasmids may be maintained in an altered form after undergoing structural changes (i.e. structural instability). The initial modification might be a rare event and fail to be detected in the absence of a growth advantage that the modified cells may exhibit over the parental strain. In fact, the extreme stability of certain natural plasmids can be explained by the fact that these plasmids do not impose a growth disadvantage on the host, 16 rather than by assuming segrega© 1990 Butterworth Publishers
M e d i u m c o m p o s i t i o n a n d m a i n t e n a n c e o f r e c o m b i n a n t p l a s m i d : Y. S h o m a n a n d A. L. D e m a i n
tional and/or structural stability. The culture stability of a recombinant plasmid, and thus its applicability for a given recombinant DNA process, depends on the following two parameters: (l) the frequency of the initial segregational or structural event and (2) the ratio of growth rates between parental and modified cells. There are numerous reports describing instability of recombinant plasmids in Bacillus, and it is a popular notion that plasmids are less stable in B. subtilis than in Escherichia coli. T M Many of the cloning vehicles constructed for B. subtilis are not structurally stable. 17-23Hybrid vectors capable of replication in E. coli and B. subtilis are known to replicate stably in E. coli, but suffer extensive deletions in B. subtilis. Ehrlich et a118 have postulated that the instability may be due to the existence, in B. subtilis, of a very efficient recombination system which is active on sequences with only a minimum degree of homology. The hypothesis that recombination is more efficient in B. subtilis than in E. coli is supported by the observation that plasmids with internal homologies 200-2000 base pairs in length are maintained quite stably in E. coli, whereas 90% of the plasmids undergo deletions with 20 generations in B. subtilis, is The basis for the cloning difficulties in B. subtilis is not well understood, but certain unique characteristics of the cloning systems in Bacillus may play a role: (1) DNA transformation in competent cells involves the entrance of single-stranded DNA into cells. Singlestranded DNA is highly susceptible to recombination with the chromosome. The result is that cloning homologous DNA in a Rec + host will give a large background of transformants produced by chromosomal integration. (2) Deletions can occur during the transformation process. Ostroff and Pene 24 proposed that competent B. subtilis strains are able to recognize and damage heterologously propagated DNA sequences. The authors showed that: (a) deletions in chimeric plasmids are targeted toward insert sequences; (b) only a single form of DNA (intact or deleted) is found in individual transformants; (c) identical chimeras isolated from E. coli, but not from B. subtilis, are damaged during transformation; (d) a spontaneous mutant that showed stable transformation had a reduced endonucleolytic degradation activity of extracellular heterologously propagated chimeric plasmid DNA. (3) Unlike E. coli, natural resistance plasmids have not been detected in B. subtilis strains. B. subtilis strain 168, which is the most common host for genetic studies, has no plasmid. 25Most of the cloning vectors for B. subtilis are chimeric plasmids from other Gram-positive strains. The present study addresses itself to the question of whether manipulation of nutrition via the composition of the growth medium can aid in the stabilization of a structurally unstable plasmid.
Materials and methods B a c t e r i a l strains a n d p l a s m i d s B. subtilis BR151 (equivalent to BGSC 1A40) was obtained from A. L. Sonenshein of Tufts University
EcoRI
•\
Ava I BamHI
,.
EcoRI ~,AkN
tms
~.
~
tl' "~' HInDIII
AvaI
Figure 1 Schematic representation of plasmid pCED3. Dark area represents pBR322 DNA: dotted areas represent pUB110 from S. aureus and the B. subtilis tins DNA; plain line represents the E. coil lacZ gene
Health Sciences Center, Boston. Its genotype is trpC2,1ys-3,metBlO. Plasmid pUBII0 is a 4.5-kb Staphylococcus attreus plasmid, encoding kanamycin (Km) 26 and bleomycin (Ble) 27 resistance, which is stably maintained in B. subtilis. 2s The 13.5-kb plasmid pCED329 was constructed from pBR322 and pUBl l0 and contains the E. coli/3-galactosidase structural gene attached to the B. subtilis tms promoter (Figure 1). Plasmid pCED3 confers /3-galactosidase activity and Km resistance in B. subtilis, while in E. coli the plasmid also encodes ampicillin (Ap) resistance. The source of the plasmids was A. L. Sonenshein. Media Complex medium: TSB is tryptic soy broth (Gibco) 30 g l -~. Defined media: these all contained inorganic salts (in g I-1: K2HPO4, 6; KHEPO 4, 2; NHaCI, l; NH4NO3, 0.2; NazSO 4, 0.2; MgSOa.7H20 , 0.02; MnSO4.4H20 , 0.002; FeSOa.7H20, 0.002; CaCI2, 0.001). In addition, they contained the following amino acids and carbon sources (in g I 1): 6AA medium: arginine.HCl, 1; aspartic acid 2; glycine, 0.4; methionine, 1; tryptophan, 1; lysine.HCl, 1; starch, 25; FD/TL medium: 6AA medium + glycerol, 2.5; 18AA medium; alanine, 0.63; glycine, 0.35; arginine.HC1, 0.9; histidine.HCI.HzO, 0.75; lysine.HCl, 1.9; proline, 0.9; serine, 0.9; threonine, 0.75; aspartic acid, 13; glutamic acid, 4.1; isoleucine, 1.1 ; leucine, 1.8; methionine, 0.45; phenylalanine, 0.55; valine, 1.3; tyrosine, 0.63; tryptophan, I; (NH4)2SO4, 0.83; D-glucosamine.HCl, 0.028; glucose, 15. The pH of the media was adjusted to 7.0 with NaOH before autoclaving. Glucose and glycerol were autoclaved separately. Kanamycin was added at 5/~g mland ampicillin at 25/~g ml- i. Solidified media contained 2% agar. E n z y m e M i c r o b . Technol., 1990, vol. 12, M a y
331
Papers Growth rate m e a s u r e m e n t s B. subtilis cultures were stored on Schaeffer sporulation agar. 3° Inocula were prepared by streaking spores from the sporulation agar onto TSB + Km or FD/ TL or Km agar plates containing X-gal (see below). Following 18 h incubation at 37°C, single blue colonies were resuspended in medium at a cell density of approximately 106 cells ml 1. Cells were placed inside side-arm flasks (250 ml) with 20 ml medium and incubated with shaking at 250 rev min J (New Brunswick, Psycrotherm Controlled Environment Incubator Shaker) at the given temperatures. Growth was determined by measuring the absorbance of the cultures (between 5 and 100 Klett units) in a Klett Summerson photoelectric colorimeter (model 800-3) using a red filter (No. 66). At least three independent measurements were done to determine the growth rates of the strains under the given conditions. Absorbance correlated with viable counts.
100
I
0
50
LLI
0 o~
10
m
M.
0
5
i 0
M e a s u r e m e n t o f p l a s m i d stability Stability of plasmids was assayed by plotting the fraction of cells bearing the plasmid-encoded lacZ + or Km r phenotype in a given population as a function of number of generations. Cells were taken from TSB + Km or FD/TL + K m agar plates that contained 40 mg 1- J of 5-bromo-4-chloro-3-indolyl-fl-o-galactopyranoside (Xgal) and were grown in flasks as above. Every 6 to 8 h, samples were diluted (1 : 500 or 1 : 1000) into fresh medium and simultaneously diluted in dilution buffer (25 mM phosphate buffer, pH 7 containing 10 -3 mM MnSO4) and plated on agar plates. Cultures that were grown in kanamycin-free medium were plated on TSB agar and the colonies transferred onto TSB + Km + X-gal agar plates. Cultures grown in kanamycincontaining medium were plated directly onto TSB + Km + X-gal agar plates. At least 100 colonies were tested, and the results were plotted as the percentage of cells of the scored phenotype (LacZ + or Km r) in the cell population as a function of the number of generations. Results
Stability o f plasmids in p U B l l O and pCED3 The model system in this study consists of the B. subtilis BR151 strain and the 13.5-kb plasmid pCED3fl 9 Several properties of pCED3 make it a good model system for the study of plasmid instability in B. subtilis: (1) Plasmid pCED3 is composed of the S. aureus high copy number plasmid pUB 110.31'32 Plasmid pUB 110 is widely used in B. subtilis cloning vectors and in E. coli-B, subtilis bifunctional vectors. The plasmid contains a kanamycin resistance gene that is expressed in both B. subtilis and E. coli. (2) Plasmid pCED3 contains a part of the E. coli replicon pBR322 enabling it to replicate in E. coll. Plasmid pBR322 is engineered into most of the B. subtilis-E, coli bifunctional plasmids used today. The bifunctional property of plasmid pCED3 allows for the direct comparison of plasmid
332
Enzyme Microb. Technol., 1990, vol. 12, May
I 10
t
I 20
NUMBER OF GENERATIONS Figure 2 Stability of the Km r phenotype (encoded by pCED3) of B. subtilis BR151 (pCED3) cultures during growth in TSB (0), and
TSB + Km (©)
stability in B. subtilis versus E. coli. (3) Part of plasmid pCED3 contains the E. coli fl-galactosidase structural gene attached to the B. subtilis tms promoter. The gene represents a "cloned foreign gene" whose product can be quantitatively assayed for enzymatic activity or qualitatively detected in colonies on agar plates containing an indicator such as X-gal. The initial experiments were on the stability of plasmid pCED3 with and without kanamycin selection. The stability of plasmid pUB 110 was tested first, since this plasmid was used in the construction of pCED3 and contains the origin of replication for B. subtilis. A previously published report 28 stated that plasmid pUB 110 is stable. However, other reports describe segregationa125'33 and structural J7'2° instabilities of plasmids introduced into B. subtilis. It was important to confirm that plasmid p U B l l 0 is stable before comparing it to the unstable plasmid pCED3fl 9 B. subtilis cells harboring plasmid p U B l l 0 were grown for more than 500 generations in TSB in the absence of kanamycin selection, and then 500 colonies were tested for resistance to kanamycin. All of the colonies exhibited the Km r phenotype indicating that the plasmid is indeed stable. The stability of plasmid pCED3 in the absence of kanamycin selection was examined next. Growth of BR151 (pCED3) cells in TSB without selection resulted in the loss of the kanamycin resistance (Km r) phenotype (Figure 2). After 15 generations of growth, less than 10% of the cells were resistant to kanamycin. It should be noted, however, that even in the presence of selective pressure only about 90% of the cells exhibited resistance to kanamycin. This observation could be a result of the transfer method in which some of the
Medium composition and maintenance of recombinant plasmid: Y. Shoman and A. L. Demain 100
'
I
'
I
--
Medium
50 ¢n _J
Temperature (°C)
Doubling time (min)
37 37 37 37 30 25
135 54 30 23 37 64
6AA FD/TL 18AA TSB TS B TSB
LU
U + N
U 10 ,
Recombinant plasmid pCED3 [confers ~-galactosidase production (LacZ +) and kanamycin resistance (Km")] in Bacillus subtilis was found to be both segregationally and structurally unstable. Since many solutions to segregational instability are already available, the problem of structural instability was specifically addressed by inclusion of kanamycin in the growth media. Culture instability was found to be highest in complex and defined media supporting high growth rates. Stabilization over the duration of the experiment (40 generations) was achieved by use of a recently developed chemically defined medium supporting a lower growth rate. Slowing down growth by decreasing temperature was much less effective. A major effect of the growth medium appears to be that of decreasing the growth rate advantage held by cells with plasmid deletions over parental cells containing the intact plasmid.
Keywords: Recombinantplasmid; Bacillus subtilis; plasmid instability
Introduction Three aspects of Bacillus are of special interest for potential industrial applications: (1) Bacillus subtilis has no known pathogenic interactions with humans or animals. 1The only authentic report of human infection due toB. subtilis was in a severely compromised host, a drug addict. 2 Unlike Gram-negative organisms, the cell surface of B. subtilis is composed only of peptidoglycan and teichoic acid.3 Therefore, recombinant DNA products produced by B. subtilis would not be contaminated by lipopolysaccharide endotoxins. 4 (2)B. subtilis is able to secrete homologous and heterologous proteins .5-9 (3) Bacillus strains have many industrial applications, and systems are well developed for large-scale cultivation. ~0Their potential in commercial genetic engineering has been reviewed by Workman et al. ~ The instability of cultures containing recombinant
The present address of Dr. Shoham is the Department of Food Engineering and Biotechnology,The Technion, Haifa 32000, Israel Address reprintrequests to Dr. Demainat the FermentationMicrobiology Laboratory Department of Biology, Massachusetts Institute of Technology,Cambridge, MA 02139 Received 5 January 1989; revised 30 May 1989 330
Enzyme Microb. Technol., 1990, vol. 12, May
plasmids is a problem often encountered in applying recombinant DNA technology.~2 During growth or storage, a population of plasmid-bearing cells often segregates to give cells that are either plasmid-free or contain modified plasmids. During large-scale production of recombinant DNA products, plasmid instability directly decreases the efficiency and productivity of the fermentation process. 13,14Industrial fermentations typically involve a large number of generations (over 50), allowing ample time for modified cells to take over the population. Especially during the manufacturing of pharmaceuticals which require GMP (Good Manufacturing Practice) conditions, the drug regulatory authorities consider plasmid instability to be undesirable. 15 Two general mechanisms are responsible for plasmid instability. First, plasmids may be lost from some cells due to unequal segregation (i.e. segregational instability). Second, plasmids may be maintained in an altered form after undergoing structural changes (i.e. structural instability). The initial modification might be a rare event and fail to be detected in the absence of a growth advantage that the modified cells may exhibit over the parental strain. In fact, the extreme stability of certain natural plasmids can be explained by the fact that these plasmids do not impose a growth disadvantage on the host, 16 rather than by assuming segrega© 1990 Butterworth Publishers
M e d i u m c o m p o s i t i o n a n d m a i n t e n a n c e o f r e c o m b i n a n t p l a s m i d : Y. S h o m a n a n d A. L. D e m a i n
tional and/or structural stability. The culture stability of a recombinant plasmid, and thus its applicability for a given recombinant DNA process, depends on the following two parameters: (l) the frequency of the initial segregational or structural event and (2) the ratio of growth rates between parental and modified cells. There are numerous reports describing instability of recombinant plasmids in Bacillus, and it is a popular notion that plasmids are less stable in B. subtilis than in Escherichia coli. T M Many of the cloning vehicles constructed for B. subtilis are not structurally stable. 17-23Hybrid vectors capable of replication in E. coli and B. subtilis are known to replicate stably in E. coli, but suffer extensive deletions in B. subtilis. Ehrlich et a118 have postulated that the instability may be due to the existence, in B. subtilis, of a very efficient recombination system which is active on sequences with only a minimum degree of homology. The hypothesis that recombination is more efficient in B. subtilis than in E. coli is supported by the observation that plasmids with internal homologies 200-2000 base pairs in length are maintained quite stably in E. coli, whereas 90% of the plasmids undergo deletions with 20 generations in B. subtilis, is The basis for the cloning difficulties in B. subtilis is not well understood, but certain unique characteristics of the cloning systems in Bacillus may play a role: (1) DNA transformation in competent cells involves the entrance of single-stranded DNA into cells. Singlestranded DNA is highly susceptible to recombination with the chromosome. The result is that cloning homologous DNA in a Rec + host will give a large background of transformants produced by chromosomal integration. (2) Deletions can occur during the transformation process. Ostroff and Pene 24 proposed that competent B. subtilis strains are able to recognize and damage heterologously propagated DNA sequences. The authors showed that: (a) deletions in chimeric plasmids are targeted toward insert sequences; (b) only a single form of DNA (intact or deleted) is found in individual transformants; (c) identical chimeras isolated from E. coli, but not from B. subtilis, are damaged during transformation; (d) a spontaneous mutant that showed stable transformation had a reduced endonucleolytic degradation activity of extracellular heterologously propagated chimeric plasmid DNA. (3) Unlike E. coli, natural resistance plasmids have not been detected in B. subtilis strains. B. subtilis strain 168, which is the most common host for genetic studies, has no plasmid. 25Most of the cloning vectors for B. subtilis are chimeric plasmids from other Gram-positive strains. The present study addresses itself to the question of whether manipulation of nutrition via the composition of the growth medium can aid in the stabilization of a structurally unstable plasmid.
Materials and methods B a c t e r i a l strains a n d p l a s m i d s B. subtilis BR151 (equivalent to BGSC 1A40) was obtained from A. L. Sonenshein of Tufts University
EcoRI
•\
Ava I BamHI
,.
EcoRI ~,AkN
tms
~.
~
tl' "~' HInDIII
AvaI
Figure 1 Schematic representation of plasmid pCED3. Dark area represents pBR322 DNA: dotted areas represent pUB110 from S. aureus and the B. subtilis tins DNA; plain line represents the E. coil lacZ gene
Health Sciences Center, Boston. Its genotype is trpC2,1ys-3,metBlO. Plasmid pUBII0 is a 4.5-kb Staphylococcus attreus plasmid, encoding kanamycin (Km) 26 and bleomycin (Ble) 27 resistance, which is stably maintained in B. subtilis. 2s The 13.5-kb plasmid pCED329 was constructed from pBR322 and pUBl l0 and contains the E. coli/3-galactosidase structural gene attached to the B. subtilis tms promoter (Figure 1). Plasmid pCED3 confers /3-galactosidase activity and Km resistance in B. subtilis, while in E. coli the plasmid also encodes ampicillin (Ap) resistance. The source of the plasmids was A. L. Sonenshein. Media Complex medium: TSB is tryptic soy broth (Gibco) 30 g l -~. Defined media: these all contained inorganic salts (in g I-1: K2HPO4, 6; KHEPO 4, 2; NHaCI, l; NH4NO3, 0.2; NazSO 4, 0.2; MgSOa.7H20 , 0.02; MnSO4.4H20 , 0.002; FeSOa.7H20, 0.002; CaCI2, 0.001). In addition, they contained the following amino acids and carbon sources (in g I 1): 6AA medium: arginine.HCl, 1; aspartic acid 2; glycine, 0.4; methionine, 1; tryptophan, 1; lysine.HCl, 1; starch, 25; FD/TL medium: 6AA medium + glycerol, 2.5; 18AA medium; alanine, 0.63; glycine, 0.35; arginine.HC1, 0.9; histidine.HCI.HzO, 0.75; lysine.HCl, 1.9; proline, 0.9; serine, 0.9; threonine, 0.75; aspartic acid, 13; glutamic acid, 4.1; isoleucine, 1.1 ; leucine, 1.8; methionine, 0.45; phenylalanine, 0.55; valine, 1.3; tyrosine, 0.63; tryptophan, I; (NH4)2SO4, 0.83; D-glucosamine.HCl, 0.028; glucose, 15. The pH of the media was adjusted to 7.0 with NaOH before autoclaving. Glucose and glycerol were autoclaved separately. Kanamycin was added at 5/~g mland ampicillin at 25/~g ml- i. Solidified media contained 2% agar. E n z y m e M i c r o b . Technol., 1990, vol. 12, M a y
331
Papers Growth rate m e a s u r e m e n t s B. subtilis cultures were stored on Schaeffer sporulation agar. 3° Inocula were prepared by streaking spores from the sporulation agar onto TSB + Km or FD/ TL or Km agar plates containing X-gal (see below). Following 18 h incubation at 37°C, single blue colonies were resuspended in medium at a cell density of approximately 106 cells ml 1. Cells were placed inside side-arm flasks (250 ml) with 20 ml medium and incubated with shaking at 250 rev min J (New Brunswick, Psycrotherm Controlled Environment Incubator Shaker) at the given temperatures. Growth was determined by measuring the absorbance of the cultures (between 5 and 100 Klett units) in a Klett Summerson photoelectric colorimeter (model 800-3) using a red filter (No. 66). At least three independent measurements were done to determine the growth rates of the strains under the given conditions. Absorbance correlated with viable counts.
100
I
0
50
LLI
0 o~
10
m
M.
0
5
i 0
M e a s u r e m e n t o f p l a s m i d stability Stability of plasmids was assayed by plotting the fraction of cells bearing the plasmid-encoded lacZ + or Km r phenotype in a given population as a function of number of generations. Cells were taken from TSB + Km or FD/TL + K m agar plates that contained 40 mg 1- J of 5-bromo-4-chloro-3-indolyl-fl-o-galactopyranoside (Xgal) and were grown in flasks as above. Every 6 to 8 h, samples were diluted (1 : 500 or 1 : 1000) into fresh medium and simultaneously diluted in dilution buffer (25 mM phosphate buffer, pH 7 containing 10 -3 mM MnSO4) and plated on agar plates. Cultures that were grown in kanamycin-free medium were plated on TSB agar and the colonies transferred onto TSB + Km + X-gal agar plates. Cultures grown in kanamycincontaining medium were plated directly onto TSB + Km + X-gal agar plates. At least 100 colonies were tested, and the results were plotted as the percentage of cells of the scored phenotype (LacZ + or Km r) in the cell population as a function of the number of generations. Results
Stability o f plasmids in p U B l l O and pCED3 The model system in this study consists of the B. subtilis BR151 strain and the 13.5-kb plasmid pCED3fl 9 Several properties of pCED3 make it a good model system for the study of plasmid instability in B. subtilis: (1) Plasmid pCED3 is composed of the S. aureus high copy number plasmid pUB 110.31'32 Plasmid pUB 110 is widely used in B. subtilis cloning vectors and in E. coli-B, subtilis bifunctional vectors. The plasmid contains a kanamycin resistance gene that is expressed in both B. subtilis and E. coli. (2) Plasmid pCED3 contains a part of the E. coli replicon pBR322 enabling it to replicate in E. coll. Plasmid pBR322 is engineered into most of the B. subtilis-E, coli bifunctional plasmids used today. The bifunctional property of plasmid pCED3 allows for the direct comparison of plasmid
332
Enzyme Microb. Technol., 1990, vol. 12, May
I 10
t
I 20
NUMBER OF GENERATIONS Figure 2 Stability of the Km r phenotype (encoded by pCED3) of B. subtilis BR151 (pCED3) cultures during growth in TSB (0), and
TSB + Km (©)
stability in B. subtilis versus E. coli. (3) Part of plasmid pCED3 contains the E. coli fl-galactosidase structural gene attached to the B. subtilis tms promoter. The gene represents a "cloned foreign gene" whose product can be quantitatively assayed for enzymatic activity or qualitatively detected in colonies on agar plates containing an indicator such as X-gal. The initial experiments were on the stability of plasmid pCED3 with and without kanamycin selection. The stability of plasmid pUB 110 was tested first, since this plasmid was used in the construction of pCED3 and contains the origin of replication for B. subtilis. A previously published report 28 stated that plasmid pUB 110 is stable. However, other reports describe segregationa125'33 and structural J7'2° instabilities of plasmids introduced into B. subtilis. It was important to confirm that plasmid p U B l l 0 is stable before comparing it to the unstable plasmid pCED3fl 9 B. subtilis cells harboring plasmid p U B l l 0 were grown for more than 500 generations in TSB in the absence of kanamycin selection, and then 500 colonies were tested for resistance to kanamycin. All of the colonies exhibited the Km r phenotype indicating that the plasmid is indeed stable. The stability of plasmid pCED3 in the absence of kanamycin selection was examined next. Growth of BR151 (pCED3) cells in TSB without selection resulted in the loss of the kanamycin resistance (Km r) phenotype (Figure 2). After 15 generations of growth, less than 10% of the cells were resistant to kanamycin. It should be noted, however, that even in the presence of selective pressure only about 90% of the cells exhibited resistance to kanamycin. This observation could be a result of the transfer method in which some of the
Medium composition and maintenance of recombinant plasmid: Y. Shoman and A. L. Demain 100
'
I
'
I
--
Medium
50 ¢n _J
Temperature (°C)
Doubling time (min)
37 37 37 37 30 25
135 54 30 23 37 64
6AA FD/TL 18AA TSB TS B TSB
LU
U + N
U 10 ,
