JOURNAL
OF INVERTEBRATE
PATHOLOGY
57,
Construction of Tracer Utilizing the xylf L.D. Department
66-70 (1991)
Plasmids for Bacillus sphaericus Gene from Pseudomonas putida
TAYLORAND W.F.
of Microbiology,
Arizona
State
1593
BURKE, JR.
University.
Tempe,
Arizona
85287-2701
Received January 29, 1990; accepted May 4, 1990 Genetically engineered microorganisms (GEMS) released into the environment must be traceable in order to accurately assess their impact on the area of release. Tracer genes other than those that introduce antibiotic resistance are preferred for use in identifying genetically engineered strains. In this study, we describe the construction of a series of tracer plasmids for use in Bacillus sphaericus using the xylE gene from the Pseudomonas putida TOL plasmid. This gene codes for the enzyme catechol 2,3-dioxygenase which converts the colorless substance catechol to 2hydroxymuconic semialdehyde, a yellow product which is easily detected. Colonies of cells which express the xylE gene turn yellow shortly after being exposed with a solution of catechol. 6 19% Academic KEY
Press, Inc. WORDS:
Bacillus
2,3-oxygenase; pUBll0.
sohaericus:
tracer vlasmids; GEMS; Pseudomonas
INTRODUCTION
xylE;
catechol
muconic semialdehyde, permitting detection within seconds. The xylE gene was used to construct three first-generation tracer plasmids with different properties which replicate and are expressed in B. sphaericus. Two are shuttle vectors capable of replicating in both Escherichia coli and Bacillus species, while the third replicates in Bacillus only.
Bacillus sphaericus has been of increasing interest due to its ability to produce a parasporal crystalline body during sporulation which is toxic to mosquito larvae (Payne and Davidson, 1984). It has proven to be an effective mosquitocidal agent which provides a viable alternative to environmentally damaging chemical pesticides. Genetic engineering of B. sphaericus to create improved variants holds the potential of making it more useful; however, before these organisms can be released into the environment, methods must be developed for the detection and enumeration of the introduced organism in order to assess its risk to the environment. Tracer genes which do not introduce drug resistance can be safely used to trace the released organism in the field. The Pseudomonas putida TOL plasmid gene xylE, which codes for the enzyme catecho1 2,3-dioxygenase (CatO,ase), produces a chromogenic change which can be used to identify an organism carrying this enzyme. Colonies of cells which express the xylE gene are able to convert a solution of the colorless and inexpensive substance catechol to the yellow product 2-hydroxy-
MATERIALS AND METHODS Bacterial strains and plasmids. The bac-
terial strains and plasmids used or constructed in this study are listed in Table 1. The B. sphaericus strains are derivatives of strain 1593 obtained from E. W. Davidson. Plasmid isolation and purification. Cells were grown in MM2 medium (3.5% antibiotic medium No. 3, Difco Laboratories; 0.5% yeast extract) with neomycin (5 kg/ml) or tetracycline (15 p&ml) added for selection. Plasmid DNA was isolated using the sodium dodecyl sulfate-NaCl method described by Gryczan et al. (1978) as modified by McDonald and Burke (1982) and purified by cesium chlorideethidium bromide equilibrium centrifugation using a Beckman L8-70M ultracentrifuge. Rapid isolation of plasmid DNA was performed as described by Anderson and McKay (1983). 66
0022-2011/91 $1.50 Copyright All rights
putida
’
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
xylE
67
TRACER PLASMIDS TABLE
1
BACTERIALSTRAINSANDPLASMIDS
Plasmid
strain Escherichia
coli
DH-1 C600
EHK3555 ASBl122 ASB1123 Bacillus
pTG402 pww53-3555 pLDTlO5 pLDT117
Hanahan (1983) Zukowski et al. (1983) Keil et al. (1985) Transformant of DH- 1, this study Transformant of DH-1, this study
pBC16 pUBll0 pLDT103
Spizizen Dubnau et al. (1%9) Dubnau Transformant of BR151, this study
pLDTll7 pLDTlO5 pLDT103
hsdR-2, thy-l; Taylor and Burke Transformant of ASB13092, this Transformant of ASB13092, this Transformant of ASB13092, this
subtilis
BR151 BD170 BD366 ASB575 Bacillus
Comments, source, or reference
sphaericus
ASB13092 ASB13208 ASB13209 ASB13212
Plasmid transformation. Introduction of plasmid DNA into E. coli cells was performed as described by Hanahan (1983). Antibiotic-resistant transformants were directly selected for on tryptose blood agar base medium (Difco) with 0.5% glucose and 50 &ml thymine (TBAB-G). Competent Bacillus subtilis cells were prepared and transformed using the method of Anagnostopoulos and Spizizen (1961). Antibiotic-resistant transformants were again selected for an TBAB-G. Transformation of B. sphaericus protoplasts was performed as described by Taylor and Burke (1989). DNA manipulations. Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs, Inc., and were used in accordance with the instructions of the manufacturer. Plasmid DNA and restriction endonuclease cleavage products were analyzed by electrophoresis through 1% horizontal agarose gels. Plasmid stability. The B. sphaericus strain to be tested for stability was grown in 10 ml of MM2 with 50 p&ml thymine at 37°C with shaking at 250 rpm. When cells reached log phase, a 50-~1 aliquot was transferred to 200 ml of prewarmed MM2 with thymine and a viable count was taken at this time. A second viable count was taken
(1989) study study study
30 min later to confirm that the cells were in log phase. A Klett-Summerson calorimeter equipped with a No. 66 red filter was used to monitor increases in cell density. Cells were grown to a Klett of 80 (approximately 1 x lo8 viable cells/ml), and dilutions were plated onto TBAB-G. Plates were incubated at 30°C for 24 hr before taking a viable count and replica plating onto medium containing antibiotic. The number of generations was calculated, and the colonies were screened for catechol2,3-dioxygenase activity by spraying with a 0.5 M solution of catechol. RESULTS Construction of pLDT103. It has been shown previously that the xylE gene from pWW0 and pWW53 is wholly contained within a 2.0-kb fragment that is bounded by a BamHI site at one extremity and an XhoI site at the other. This fragment was cloned into the E. coli plasmid vector pKT230 to create the plasmid pWW53-3555 by Keil et al. (1985). A 3.1-kb BamHI fragment, carrying the xylE gene, from pWW53-3555 was isolated by electroelution from an agarose gel and ligated into the BamHI site of pUBll0. The resulting plasmid, pLDT103 (Fig. l), was transformed into B. subtilis
TAYLOR
68 BamHI PVUII pLDT103
EcoRI
1
pLDT105
(9.3kb)
EcoRI I
HpaI
PstI I 4 AP’
l3
EcoRI
B 111
h-!
6-l
:6
xylE
EeoRI I %
pLDT117
I 5
BamHI Xhol I
or,
0
BamHI PVUII
BamHI I 4
1 3
2 NmT
BamHI
Hind111
XbaI BglII I
(7.6kb) 0
AND BURKE
AvaI I 5
6
PstI
AvsI , 7
AvsI
Ave.1
BamHI KpnI I 9
BamHI KpnI
(9.3kb) 0
14&-T3
on
4 AP’
5
6
7
9 xylE
FIG. 1. Plasmid maps of pLDT103, pLDTlO5, and pLDTl17. The solid lines represent DNA from the vectors functional in Gram-positive organisms, and the serrated lines represent DNA from Gram-negative organisms. The ori indicates the orientation of the plasmid replication origin functional in Bacillus sphericus. Arrows associated with structural genes indicate direction of transcription.
and neomycin-resistant transformants were selected. Expression of xylE was detected by spraying the transformant colonies with a solution of catechol. Yellow colonies indicated the presence of CatO,ase, while cells which were unable to express xylE remained white. The B. sphaericus strain 13092 was transformed with pLDT103, and transformants were screened for CatO,ase activity by spraying with catechol. Plasmid DNA isolated from transformed strains was confirmed to be pLDT103 by restriction endonuclease digestion and electrophoretic analysis. Construction of pLDTl05. The xylE gene, carried on the 2.0-kb BamHIIXhoI fragment from pWW0, was transferred to an E. colilB. subtifis shuttle vector to create the plasmid pTG402 by Zukowski et al. (1983). However, we were unable to transform B. sphaericus with pTG402, suggesting that this plasmid is incapable of replication in B. sphaericus or that some feature of this plasmid DNA is lethal to this particular bacterial host. A 5.7-kb fragment containing the xylE
gene and the pBR322 ampicillin resistance gene and origin of replication was isolated after digestion of pTG402 with both BamHI and EcoRI. This fragment was ligated to pBC16 fragments resulting from a complete digest of the plasmid with BumHI and a partial digest with EcoRI. The ligation mixture was then used to transform E. coli DH-1 cells. Tetracycline- and ampicillin-resistant transformants were selected and screened for xylE expression and chloramphenicol sensitivity. A plasmid displaying these phenotypes was chosen at random and was designated pLDT105 (Fig. 1). DNA from a rapid plasmid isolation was used to transform B. sphaericus 13092 cells. Tetracycline-resistant transformants were selected and sprayed with catechol to screen for CatO,ase activity. The anticipated plasmid map was confirmed by restriction analysis. Construction of pLDT117. The plasmid vector pLDT117 (Fig. 1) is similar to pLDT105, except that the origin of replication and the antibiotic resistance functional in B. sphaericus are from the Staphylococcus aureus plasmid pUBll0. This plasmid
TRACER PLASMIDS
xylE
was constructed by digesting pTG402 and PUB 110 with BamHI and EcoRI. The fragments were ligated and the mixture was used to transform E. coli DH-1. Neomycinand ampicillin-resistant transformants were selected and screened for xylE expression and chloramphenicol sensitivity. One plasmid isolated from a transformant was designated pLDTll7 and was mapped with restriction endonucleases. This plasmid was used to transform B. sphaericus 13092 and was found to exhibit CatO,ase activity. Plasmids isolated from these transformants were physical mapped with restriction endonucleases and were confirmed to be pLDTll7. Plasmid stability. Derivatives of B. sphaericus 1593 containing the plasmids pLDT103, pLDT105, and pLDT117 were tested for their ability to stably maintain the plasmids in the absence of selective pressure. The results are shown in Table 2. Only one strain carrying pLDT103 was stably maintained without selection. DISCUSSION
The three tracer plasmids constructed in this study are capable of replicating and expressing the xylE gene in the Gram-negative E. coli and two species of Gram-positive Bacillus, including B. sphaericus. The presence of these plasmids in GEMS, together with other phenotypic characteristics such as naturally occurring antibiotic resistances, provides a rapid and unequivocal means of identification. An important feature in successfully tracing a genetically manipulated microorganTABLE PLASMID
STABILITY
2
IN Bacillus
sphaericus
Percentage of colonies maintaining Plasmid
No. of generations
pLDT103 pLDTlO5 pLDTll7
11.3 11.6 12.3
Antibiotic 99.5 9.2 16.1
CatO,ase activity 92.3 8.6 15.5
69
ism is the stability of the tracer gene within the strain. In B. sphaericus, stability varied considerably from plasmid to plasmid. The smaller, nonshuttle vector plasmid, pLDT103 (7.6 kb), was very stable; however, the two slightly larger plasmids, pLDTlO5 (9.3 kb) and pLDT117 (9.3 kb), showed poor stability. Size-dependent instability was shown to be a common property of PUB 110 plasmids in B. subtilis, with plasmids carrying inserts larger than 3-4 kb being poorly maintained (Bron et al., 1988). As pBC16 shares considerable homology with pUBl10, the instability of these larger tracer plasmids in B. sphaericus might also be related to plasmid size. The pUB1 lobased plasmid, pLDT117, was slightly more stable than the pBC16-based plasmid, pLDT105, in B. sphaericus. This property was also observed by McDonald and Burke (1984). However, CatO,ase activity in pLDT105 was higher than in either pLDT103 or pLDT117. The cloned xylE gene does not have a functional promoter in Bacillus species and, therefore, must come under the control of an existing promoter within the vector. The CatO,ase activity observed in the plasmids would suggest that the tetracycline-resistance gene promoter of pBC16, which presumably controls transcription of the xylE gene via readthrough, is a stronger promoter than that of the neomycin-resistance gene of PUB 110. Phenotypic tracers are becoming increasingly important as greater numbers of genetically engineered organisms are examined for release into the environment. Such tracer genes must be maintained on highly stable plasmids or integrated into the chromosome of the host organism. Plasmids such as pLDT103 or other even more stable derivatives which lack antibiotic-resistance genes hold the promise of effective tracer systems for use in the extremely important entomocidal organism, B. sphaericus 1593. ACKNOWLEDGMENTS This investigation received financial support from
70
TAYLOR
the UNDPiWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases and from the Arizona Disease Control Research Commission. In addition, support was provided in part by Biomedical Research Grant S07RR07112 awarded to Arizona State University by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health.
REFERENCES ANAGNOSTOPOULOS, C., AND SPIZIZEN. J. 1961. Requirements for transformation in Bacillus subtilis. J. Bacterial.,
81, 741-746.
ANDERSON, D. G., AND MCKAY, L. L. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbial., 46, 549-552. BRON, S., LUXEN, E., AND SWART, P. 1988. Instability of recombinant pUBll0 plasmids in Bacillus subtilis: Plasmid-encoded stability function and effects of DNA inserts. Plasmid, 19, 231-241. DUBNAU, D., DAVIDOFF-ABELSON, R., AND SMITH, I. 1969. Transformation and transduction in Bacillus subtilis: Evidence for separate modes of recombination formation. J. Mol. Biol., 45, 155-179. GRYCZAN, T. J., CONTENTE, S., AND DUBNAU, D. 1978. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J. Bacterial., 134, 318-329.
AND BURKE HANAHAN, D. 1983. Studies on the transformation of Escherichiu coli with plasmids. J. Mol. Biol., 166, 557-580. KEIL, H., KEIL, S., PICKUP, R. W., AND WILLIAMS, P. A. 1985. Evolutionary conservation of genes coding for meta pathway enzymes within TOL plasmids pWW0 and pWW53. J. Bacterial.. 164, 887-895. MCDONALD, K. 0.. AND BURKE, W. F., JR. 1982. Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis. J. Bacterial., 149, 391-394. MCDONALD, K. O., AND BURKE, W. F., JR. 1984. Plasmid transformation of Bacillus sphaericus 1593. J. Gen. Microbial., 130, 203-208. PAYNE, J., AND DAVIDSON, E. W. 1984. Insecticidal activity of the crystalline parasporal inclusions and other components of the Bacillus sphaericus 1593 spore complex. J. Invertebr. Pathol., 43, 383-388. TAYLOR, L. D., AND BURKE, W. F., JR. 1989. Improved procedure for the transformation of the entomopathic microorganism Bacillus sphaericus. J. Microbial.
Methods,
9, 35-39.
ZUKOWSKI, M. M., GAFFNEY, D. F., SPECK, D., KAUFFMANN, M., FINDELLI, A., WISECUP, A., AND LECOCQ, J.-P. 1983. Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc. Natl. Acad. Sci. USA, 80, 1101-1105.
OF INVERTEBRATE
PATHOLOGY
57,
Construction of Tracer Utilizing the xylf L.D. Department
66-70 (1991)
Plasmids for Bacillus sphaericus Gene from Pseudomonas putida
TAYLORAND W.F.
of Microbiology,
Arizona
State
1593
BURKE, JR.
University.
Tempe,
Arizona
85287-2701
Received January 29, 1990; accepted May 4, 1990 Genetically engineered microorganisms (GEMS) released into the environment must be traceable in order to accurately assess their impact on the area of release. Tracer genes other than those that introduce antibiotic resistance are preferred for use in identifying genetically engineered strains. In this study, we describe the construction of a series of tracer plasmids for use in Bacillus sphaericus using the xylE gene from the Pseudomonas putida TOL plasmid. This gene codes for the enzyme catechol 2,3-dioxygenase which converts the colorless substance catechol to 2hydroxymuconic semialdehyde, a yellow product which is easily detected. Colonies of cells which express the xylE gene turn yellow shortly after being exposed with a solution of catechol. 6 19% Academic KEY
Press, Inc. WORDS:
Bacillus
2,3-oxygenase; pUBll0.
sohaericus:
tracer vlasmids; GEMS; Pseudomonas
INTRODUCTION
xylE;
catechol
muconic semialdehyde, permitting detection within seconds. The xylE gene was used to construct three first-generation tracer plasmids with different properties which replicate and are expressed in B. sphaericus. Two are shuttle vectors capable of replicating in both Escherichia coli and Bacillus species, while the third replicates in Bacillus only.
Bacillus sphaericus has been of increasing interest due to its ability to produce a parasporal crystalline body during sporulation which is toxic to mosquito larvae (Payne and Davidson, 1984). It has proven to be an effective mosquitocidal agent which provides a viable alternative to environmentally damaging chemical pesticides. Genetic engineering of B. sphaericus to create improved variants holds the potential of making it more useful; however, before these organisms can be released into the environment, methods must be developed for the detection and enumeration of the introduced organism in order to assess its risk to the environment. Tracer genes which do not introduce drug resistance can be safely used to trace the released organism in the field. The Pseudomonas putida TOL plasmid gene xylE, which codes for the enzyme catecho1 2,3-dioxygenase (CatO,ase), produces a chromogenic change which can be used to identify an organism carrying this enzyme. Colonies of cells which express the xylE gene are able to convert a solution of the colorless and inexpensive substance catechol to the yellow product 2-hydroxy-
MATERIALS AND METHODS Bacterial strains and plasmids. The bac-
terial strains and plasmids used or constructed in this study are listed in Table 1. The B. sphaericus strains are derivatives of strain 1593 obtained from E. W. Davidson. Plasmid isolation and purification. Cells were grown in MM2 medium (3.5% antibiotic medium No. 3, Difco Laboratories; 0.5% yeast extract) with neomycin (5 kg/ml) or tetracycline (15 p&ml) added for selection. Plasmid DNA was isolated using the sodium dodecyl sulfate-NaCl method described by Gryczan et al. (1978) as modified by McDonald and Burke (1982) and purified by cesium chlorideethidium bromide equilibrium centrifugation using a Beckman L8-70M ultracentrifuge. Rapid isolation of plasmid DNA was performed as described by Anderson and McKay (1983). 66
0022-2011/91 $1.50 Copyright All rights
putida
’
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
xylE
67
TRACER PLASMIDS TABLE
1
BACTERIALSTRAINSANDPLASMIDS
Plasmid
strain Escherichia
coli
DH-1 C600
EHK3555 ASBl122 ASB1123 Bacillus
pTG402 pww53-3555 pLDTlO5 pLDT117
Hanahan (1983) Zukowski et al. (1983) Keil et al. (1985) Transformant of DH- 1, this study Transformant of DH-1, this study
pBC16 pUBll0 pLDT103
Spizizen Dubnau et al. (1%9) Dubnau Transformant of BR151, this study
pLDTll7 pLDTlO5 pLDT103
hsdR-2, thy-l; Taylor and Burke Transformant of ASB13092, this Transformant of ASB13092, this Transformant of ASB13092, this
subtilis
BR151 BD170 BD366 ASB575 Bacillus
Comments, source, or reference
sphaericus
ASB13092 ASB13208 ASB13209 ASB13212
Plasmid transformation. Introduction of plasmid DNA into E. coli cells was performed as described by Hanahan (1983). Antibiotic-resistant transformants were directly selected for on tryptose blood agar base medium (Difco) with 0.5% glucose and 50 &ml thymine (TBAB-G). Competent Bacillus subtilis cells were prepared and transformed using the method of Anagnostopoulos and Spizizen (1961). Antibiotic-resistant transformants were again selected for an TBAB-G. Transformation of B. sphaericus protoplasts was performed as described by Taylor and Burke (1989). DNA manipulations. Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs, Inc., and were used in accordance with the instructions of the manufacturer. Plasmid DNA and restriction endonuclease cleavage products were analyzed by electrophoresis through 1% horizontal agarose gels. Plasmid stability. The B. sphaericus strain to be tested for stability was grown in 10 ml of MM2 with 50 p&ml thymine at 37°C with shaking at 250 rpm. When cells reached log phase, a 50-~1 aliquot was transferred to 200 ml of prewarmed MM2 with thymine and a viable count was taken at this time. A second viable count was taken
(1989) study study study
30 min later to confirm that the cells were in log phase. A Klett-Summerson calorimeter equipped with a No. 66 red filter was used to monitor increases in cell density. Cells were grown to a Klett of 80 (approximately 1 x lo8 viable cells/ml), and dilutions were plated onto TBAB-G. Plates were incubated at 30°C for 24 hr before taking a viable count and replica plating onto medium containing antibiotic. The number of generations was calculated, and the colonies were screened for catechol2,3-dioxygenase activity by spraying with a 0.5 M solution of catechol. RESULTS Construction of pLDT103. It has been shown previously that the xylE gene from pWW0 and pWW53 is wholly contained within a 2.0-kb fragment that is bounded by a BamHI site at one extremity and an XhoI site at the other. This fragment was cloned into the E. coli plasmid vector pKT230 to create the plasmid pWW53-3555 by Keil et al. (1985). A 3.1-kb BamHI fragment, carrying the xylE gene, from pWW53-3555 was isolated by electroelution from an agarose gel and ligated into the BamHI site of pUBll0. The resulting plasmid, pLDT103 (Fig. l), was transformed into B. subtilis
TAYLOR
68 BamHI PVUII pLDT103
EcoRI
1
pLDT105
(9.3kb)
EcoRI I
HpaI
PstI I 4 AP’
l3
EcoRI
B 111
h-!
6-l
:6
xylE
EeoRI I %
pLDT117
I 5
BamHI Xhol I
or,
0
BamHI PVUII
BamHI I 4
1 3
2 NmT
BamHI
Hind111
XbaI BglII I
(7.6kb) 0
AND BURKE
AvaI I 5
6
PstI
AvsI , 7
AvsI
Ave.1
BamHI KpnI I 9
BamHI KpnI
(9.3kb) 0
14&-T3
on
4 AP’
5
6
7
9 xylE
FIG. 1. Plasmid maps of pLDT103, pLDTlO5, and pLDTl17. The solid lines represent DNA from the vectors functional in Gram-positive organisms, and the serrated lines represent DNA from Gram-negative organisms. The ori indicates the orientation of the plasmid replication origin functional in Bacillus sphericus. Arrows associated with structural genes indicate direction of transcription.
and neomycin-resistant transformants were selected. Expression of xylE was detected by spraying the transformant colonies with a solution of catechol. Yellow colonies indicated the presence of CatO,ase, while cells which were unable to express xylE remained white. The B. sphaericus strain 13092 was transformed with pLDT103, and transformants were screened for CatO,ase activity by spraying with catechol. Plasmid DNA isolated from transformed strains was confirmed to be pLDT103 by restriction endonuclease digestion and electrophoretic analysis. Construction of pLDTl05. The xylE gene, carried on the 2.0-kb BamHIIXhoI fragment from pWW0, was transferred to an E. colilB. subtifis shuttle vector to create the plasmid pTG402 by Zukowski et al. (1983). However, we were unable to transform B. sphaericus with pTG402, suggesting that this plasmid is incapable of replication in B. sphaericus or that some feature of this plasmid DNA is lethal to this particular bacterial host. A 5.7-kb fragment containing the xylE
gene and the pBR322 ampicillin resistance gene and origin of replication was isolated after digestion of pTG402 with both BamHI and EcoRI. This fragment was ligated to pBC16 fragments resulting from a complete digest of the plasmid with BumHI and a partial digest with EcoRI. The ligation mixture was then used to transform E. coli DH-1 cells. Tetracycline- and ampicillin-resistant transformants were selected and screened for xylE expression and chloramphenicol sensitivity. A plasmid displaying these phenotypes was chosen at random and was designated pLDT105 (Fig. 1). DNA from a rapid plasmid isolation was used to transform B. sphaericus 13092 cells. Tetracycline-resistant transformants were selected and sprayed with catechol to screen for CatO,ase activity. The anticipated plasmid map was confirmed by restriction analysis. Construction of pLDT117. The plasmid vector pLDT117 (Fig. 1) is similar to pLDT105, except that the origin of replication and the antibiotic resistance functional in B. sphaericus are from the Staphylococcus aureus plasmid pUBll0. This plasmid
TRACER PLASMIDS
xylE
was constructed by digesting pTG402 and PUB 110 with BamHI and EcoRI. The fragments were ligated and the mixture was used to transform E. coli DH-1. Neomycinand ampicillin-resistant transformants were selected and screened for xylE expression and chloramphenicol sensitivity. One plasmid isolated from a transformant was designated pLDTll7 and was mapped with restriction endonucleases. This plasmid was used to transform B. sphaericus 13092 and was found to exhibit CatO,ase activity. Plasmids isolated from these transformants were physical mapped with restriction endonucleases and were confirmed to be pLDTll7. Plasmid stability. Derivatives of B. sphaericus 1593 containing the plasmids pLDT103, pLDT105, and pLDT117 were tested for their ability to stably maintain the plasmids in the absence of selective pressure. The results are shown in Table 2. Only one strain carrying pLDT103 was stably maintained without selection. DISCUSSION
The three tracer plasmids constructed in this study are capable of replicating and expressing the xylE gene in the Gram-negative E. coli and two species of Gram-positive Bacillus, including B. sphaericus. The presence of these plasmids in GEMS, together with other phenotypic characteristics such as naturally occurring antibiotic resistances, provides a rapid and unequivocal means of identification. An important feature in successfully tracing a genetically manipulated microorganTABLE PLASMID
STABILITY
2
IN Bacillus
sphaericus
Percentage of colonies maintaining Plasmid
No. of generations
pLDT103 pLDTlO5 pLDTll7
11.3 11.6 12.3
Antibiotic 99.5 9.2 16.1
CatO,ase activity 92.3 8.6 15.5
69
ism is the stability of the tracer gene within the strain. In B. sphaericus, stability varied considerably from plasmid to plasmid. The smaller, nonshuttle vector plasmid, pLDT103 (7.6 kb), was very stable; however, the two slightly larger plasmids, pLDTlO5 (9.3 kb) and pLDT117 (9.3 kb), showed poor stability. Size-dependent instability was shown to be a common property of PUB 110 plasmids in B. subtilis, with plasmids carrying inserts larger than 3-4 kb being poorly maintained (Bron et al., 1988). As pBC16 shares considerable homology with pUBl10, the instability of these larger tracer plasmids in B. sphaericus might also be related to plasmid size. The pUB1 lobased plasmid, pLDT117, was slightly more stable than the pBC16-based plasmid, pLDT105, in B. sphaericus. This property was also observed by McDonald and Burke (1984). However, CatO,ase activity in pLDT105 was higher than in either pLDT103 or pLDT117. The cloned xylE gene does not have a functional promoter in Bacillus species and, therefore, must come under the control of an existing promoter within the vector. The CatO,ase activity observed in the plasmids would suggest that the tetracycline-resistance gene promoter of pBC16, which presumably controls transcription of the xylE gene via readthrough, is a stronger promoter than that of the neomycin-resistance gene of PUB 110. Phenotypic tracers are becoming increasingly important as greater numbers of genetically engineered organisms are examined for release into the environment. Such tracer genes must be maintained on highly stable plasmids or integrated into the chromosome of the host organism. Plasmids such as pLDT103 or other even more stable derivatives which lack antibiotic-resistance genes hold the promise of effective tracer systems for use in the extremely important entomocidal organism, B. sphaericus 1593. ACKNOWLEDGMENTS This investigation received financial support from
70
TAYLOR
the UNDPiWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases and from the Arizona Disease Control Research Commission. In addition, support was provided in part by Biomedical Research Grant S07RR07112 awarded to Arizona State University by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health.
REFERENCES ANAGNOSTOPOULOS, C., AND SPIZIZEN. J. 1961. Requirements for transformation in Bacillus subtilis. J. Bacterial.,
81, 741-746.
ANDERSON, D. G., AND MCKAY, L. L. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbial., 46, 549-552. BRON, S., LUXEN, E., AND SWART, P. 1988. Instability of recombinant pUBll0 plasmids in Bacillus subtilis: Plasmid-encoded stability function and effects of DNA inserts. Plasmid, 19, 231-241. DUBNAU, D., DAVIDOFF-ABELSON, R., AND SMITH, I. 1969. Transformation and transduction in Bacillus subtilis: Evidence for separate modes of recombination formation. J. Mol. Biol., 45, 155-179. GRYCZAN, T. J., CONTENTE, S., AND DUBNAU, D. 1978. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J. Bacterial., 134, 318-329.
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