ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 187, No. 1, April 15, pp. 229-234, 1978
Plasmid
Transfer
and LARRY
Department
Expression MILLER
of Microbiology,
in Rhodopseudomonas AND Uniuersity
Received September
SAMUEL of Illinois,
sphaeroides’
KAPLAN Urbana,
Illinois
61801
19, 1977; revised December 9, 1977
Plasmid RP4 (among others) has been transferred from Escherichia coli to Rhodopseudomonas sphaeroides. Data bearing on the physical presence of the plasmid and its expression of drug resistance determinants in R. sphaeroides are presented. Conditions of transfer between R. sphaeroides strains, between R. sphaeroides and R. capsulata, and between R. sphaeroides and E. cob’ have been carefully defined.
Many drug resistance plasmids originally detected in Pseudomonas sp. have been shown to cross generic lines. In some cases, certain of the plasmids have been shown to effect chromosomal mobilization in the new host (1, 2). One plasmid in particular, RP4, has been used in many such experiments. A report by Olsen and Shipley (3) indicated that these authors were able to mobilize the plasmid R1822 [identical or very similar to RP4 (4)] from Pseudomonas aeruginosa into Rhodopseudomonas sphaeroides strain MK, but the new host was unable to maintain the plasmid. Further, mobilization of the plasmid in R. sphaeroides was not demonstrated. In trying to develop a genetic system in R. sphaeroides, we attempted to extend the experiments of Olsen and Shipley in the hopes of obtaining a variant of either the host or the plasmid which would result in plasmid stability and chromosomal mobilization. The work presented in this paper deals with the optimization of transfer of RP4 from Pseudomonas aeruginosa or Escherichia coli to members of the Rhodospirillaceae, between members of the Rhodospirillaceae, and back to E. coli. MATERIALS Bacterial
AND
METHODS
Strains
The strains used are listed in Table I. Media
and Growth
Escherichia ‘This National
coli
Conditions
Mutagenesis Chemotrophically grown R. sphaeroides were mutagenixed with nitrosoquanidine. Twenty milliliters of log-phase cells (5 x 108 cells/ml) was harvested by centrlfugation, washed, and resuspended in 5 ml of Tris-malate (T-M) buffer (0.2 M Trls. 0.2 M malate, pH 6.0) with 100 gg/ml of nitrosoguanidlne. The suspension was incubated for 20 min at 34°C. after which time the cells were harvested by centrifugation, washed twice in T-M buffer, and inoculated into 20 ml of PY medium. After overnight chemotrophic growth, the cells were harvested, washed twice with ice-cold SIS, and resuspended in 10 ml of SIS, and 0.5
* Abbrevations used PY, SIS, Sistrom’s minimal salts; kanamycin; Tc, tetracycline; by a grant from the streptomycin; Rif, rifampicin; ing. 229
and Pseudomonas
work was supported Science Foundation.
were routinely grown on liquid peptone-yeast extract (PYlz medium (3 g of peptone, 3 g of yeast extract/liter, pH 7.0) at 37 or 34”C, respectively. For plating, agar was added to the medium to a final concentration of 1.5% for bottom agar or 0.7% for soft agar. Rhndopseudomonas sphaeroides and Rhodopseudomonas capsulata were grown on Sistrom’n minimal salts @IS) medium (5) at 34°C. Phototrophic growth was accomplished by inoculating cells into a screwcapped tube completely filled with SIS and incubating at a light intensity of 700 fc. Phototrophic growth on liquid medium was accomplished by sealing plates in a brewer jar made anaerobic by a Gaspak (BBL, Cockeysville, Maryland) and incubating the jar at 34°C with 700 fc incident on the jar. For chemotrophic growth, cells were grown in Delong flasks filled to 20% capacity in a water bath with shaking. Media were supplemented with amino acids ‘(40 cg/ml, final concentration) when necessary to allow the growth of auxotrophs.
ooO3-9861/78/1871-0153~.00/0 Copyright 0 1978by Academic Press, I,,c. All rights of reproduction in any form reserved.
aeruginosa
peptone-yeast extract; T-M, Tris-malate; K, Cb, carbenicilhn; Sm, EOP, efficiency of plat-
MILLER
230
AND
KAPLAN
TABLE
I
BACTERIAL STRAINS USED IN THIS INWWTIGATION Strain
Plasmid
Genotypes
RP4 R6886 None PLM2
pro, met, F+ (K, Tc, Cb) thr, ku, thi, str hp (am), lac bm), [(K, Tc bm), Cb (am)1
Source
Escherichia coli 553 (RP4) 553.1 (R6886) 0500 CA274
pro, met, F+ (K, Tc, Cb)
R. W. Hedges R. W. Hedges L. Mindich
Pseudomonas aeruginosa PAL (R1822) PA025
R1822 R68.45
Prototroph (K, Tc, Cb) arg, leu (K, Tc, Cb)
R. H. Olsen J. D. Wall
Rhodopseudomonas sphaeroides 2.4.1 cu602 CU616 cm04
None None None RP4
Prototroph
trp, rib crt hp, his, rib str, crt Prototroph
(K, Tc, Cb)”
This study This study This study
Prototroph,
rif
H-C. Yen
Rhodopseudomonas capsulata SB1003 a The phenotype resistant (see text).
None
of CU604 is Cb sensitive,
although
ml was inoculated into SIS. After two doublings, sodium ampicillin was added to a final concentration of 100 ~/ml. When the optical density of the culture declined, the cells were harvested and allowed to recover in PY medium overnight. After three cycles of ampicillin enrichment, the cells were diluted and plated on SIS minimal agar supplemented with 2 ml of nutrient broth/liter. After 4 days of growth, minute colonies were picked to PY agar and tested for auxotrophy by replica plating to SIS minimal agar supplemented with amino acids.
Antibiotics All antibiotics were freshly prepared before use. With the exception of rifampicin, all were dissolved in distilled water and filter-sterilized. Rifampicin was dissolved in absolute methanol and used without further sterilization. Final concentrations of antibiotics used for E. coli and P. aeruginosa were: kanamycin (K), 100 &ml; tetracycline (Tc), 25 &ml, carbenicillin (Cb), 100 &ml. Final concentrations for R. sphaeroides and R. capsulata were: K, 20 pg/ml, Tc, 10 pg/ml; Cb, 50 pgg/ml; streptomycin (Sm) and rifampicin (Rif), 50 &ml.
Mating Techniques Liquid mating. Donor and recipient cells were grown either chemotrophically or phototrophically to 5 x l@ cells/ml, harvested, and washed 2~ in ice-cold SIS. The pellets were resuspended in SIS to a final concentration of 1 x 10’ cells/ml. One milliliter each of donor and recipient was added to a 13-mm test tube and incubated statically at 34’C for 4 h. The mixture was then diluted, and 0.1~ml aliquots of each dilution were mixed with 3.0 ml of SIS soft agar and plated on SIS agar at 34°C. After 18 h the plates were overlayed with 10 ml of SIS soft agar containing the selective
mating to E. coli indicates
that the genotype
is Cb
antibiotics to yield a final concentration in the plates as mentioned above. Plate mating. Plate mating differed from liquid mating only in that there was no 34°C incubation prior to dilution and plating.
Plasmid Isolation Plasmids were isolated from E. coli following the procedure of Meyers et al. (6). Plasmids were isolated from R. sphaeroides as follows: Cells were grown chemotrophically in 750 ml of SIS medium. The culture was sparged with a mixture of air and oxygen to ensure an oxygen content of 13 ppm. When cell density reached 1 x 10’ cells/ml, the culture was harvested by centrifugation and lysed using the method of Grinstead et al. (7) with the omission of the shearing step. The lysate was then centrifuged at 18,000 g (25 min, 2°C) and the resulting cleared lysate was subjected to the polyethylene glycol precipitation technique of Humphreys et al. (8). Agarose gel electrophoresis was carried out in the buffer system described by Greene et al. (9). 0.7% Agarose gels were cast as vertical slabs, 8 X 10 X 0.3 cm. After casting, a piece of silk was used across the bottom of the gel to prevent slippage during electrophoresis. A current of 60 mA was applied to the gels for 1 h or until the dye front was at the bottom of the gel. DNA was visualized by soaking the gel for 1 h in electrophoresis buffer containing 0.4 pg of ethidium bromide/ml and examining the gel under short-wave uv light. RESULTS
Intra- and Interspecies Transfer of Plasmid RP4 Initial experiments involving the transfer of RP4 from Escherichia coli to Rhodo-
PLASMIDS
231
IN Rhodopseudomonas sphaeroides
pseudomonas sphaeroides were performed using the liquid mating technique, followed by phototrophic growth on SIS minimal agar supplemented with kanamycin. These experiments resulted in a frequency of transfer of the selected marker of 1 x 10V6 per donor cell. Control plates showed no spontaneous resistance to kanamycin. were colonies Kanamycin-resistant picked and tested for resistance to the unselected markers Tc and Cb. All colonies tested were found to be resistant to Tc, but not Cb. These colonies were then streaked for isolation on PY agar containing K and Tc to ensure that all isolates were free of residual E. coli. After three streakings, one clone, designated CU604, was picked for further study. Since the mechanisms of action of K and Tc both involve inhibition of protein synthesis at the level of the ribosome, and since Cb resistance was not seen in any of the K, Tc-resistant cells, it was considered possible that resistance to CU604 was due to a single chromosomal mutation resulting in resistance to both drugs, rather than to the presence of the plasmid. In view of the fact that spontaneous resistance to kanamycin was not seen on control plates, the possibility was deemed unlikely, but further tests were performed to eliminate the possibility of a single-site mutation. Tests to determine the stability of the kanamycin and tetracycline resistance were performed by growing CU604 chemotrophically for a total of 20 generations in the absence of antibiotics. At the end of this time, viable counts of the culture were made on SIS, SIS + K, and SIS + Tc. The results of such testing showed that CU604 retained the kanamycin resistance marker in all cells, while the Tc marker was retained in less than 1% of the cells, a clear indication that the resistance to K was independent of Tc resistance. Subsequent tests on CU604 which had been maintained on K and Tc for 8 months indicated that both K and Tc resistances were extremely stable. To establish unequivocally that K and Tc resistance was the result of the presence of RP4, CU604 was mated to E. coli C600 and R. sphaeroides, CU602, and the result-
ing Tc-resistant transcipients were used for plasmid isolation. CU602 was used as a recipient in the intraspecies cross, since it is both Rif resistant (allowing counterselection against CU604) and green (allowing clear distinction of transcipients from the red donor). Both liquid and plate matings were performed for the intra- and interspecies crosses in an attempt to optimize mating conditions. As can be seen from Table II, the plate mating technique was more efficient as a condition for plasmid transfer, and was adopted for routine use. Additionally, Table II shows that, in the interspecies cross, all E. coli transcipients selected for Tc resistance also acquired resistance to Cb, while 78% acquired K resistance, indicating that the plasmid remains functionally intact in R. sphaeroides, although the p-lactamase activity responsible for Cb resistance apparently is not expressed or is ineffective in protecting R. sphaeroides. The fact that the plasmid remains intact is borne out by the fact that a plasmid could be isolated from both E. coli and R. sphaeroides transcipients which corn&rated on agarose gels with RP4 (Fig. 1). While R. sphaeroides 2.4.1 normally carries at least two (10) and possibly three covalently closed circular DNA species (ll), none of these con&rates with RP4. As can be seen in Fig. 1, R. sphaeroides 2.4.1 carries two plasmids whose molecular weights have been determined to be 7.5 and 2.8 x 107.E. coli C600 carries no plasmid. CU604 carries a third plasmid with a molecular weight of 4.0 X 10’ and whose presence confers kanamycin and tetracycline resistance. Kanamycin, tetracycline, and carbenicillin resistTABLE II CONDITIONS OF MATING Recipient”
cu602 cm02 C600 C600
Mating
Liquid Plats Liquid Plate
Selected marker transfer frequency per donor 1.4 x 9.8 x
Plasmid
Transfer
and LARRY
Department
Expression MILLER
of Microbiology,
in Rhodopseudomonas AND Uniuersity
Received September
SAMUEL of Illinois,
sphaeroides’
KAPLAN Urbana,
Illinois
61801
19, 1977; revised December 9, 1977
Plasmid RP4 (among others) has been transferred from Escherichia coli to Rhodopseudomonas sphaeroides. Data bearing on the physical presence of the plasmid and its expression of drug resistance determinants in R. sphaeroides are presented. Conditions of transfer between R. sphaeroides strains, between R. sphaeroides and R. capsulata, and between R. sphaeroides and E. cob’ have been carefully defined.
Many drug resistance plasmids originally detected in Pseudomonas sp. have been shown to cross generic lines. In some cases, certain of the plasmids have been shown to effect chromosomal mobilization in the new host (1, 2). One plasmid in particular, RP4, has been used in many such experiments. A report by Olsen and Shipley (3) indicated that these authors were able to mobilize the plasmid R1822 [identical or very similar to RP4 (4)] from Pseudomonas aeruginosa into Rhodopseudomonas sphaeroides strain MK, but the new host was unable to maintain the plasmid. Further, mobilization of the plasmid in R. sphaeroides was not demonstrated. In trying to develop a genetic system in R. sphaeroides, we attempted to extend the experiments of Olsen and Shipley in the hopes of obtaining a variant of either the host or the plasmid which would result in plasmid stability and chromosomal mobilization. The work presented in this paper deals with the optimization of transfer of RP4 from Pseudomonas aeruginosa or Escherichia coli to members of the Rhodospirillaceae, between members of the Rhodospirillaceae, and back to E. coli. MATERIALS Bacterial
AND
METHODS
Strains
The strains used are listed in Table I. Media
and Growth
Escherichia ‘This National
coli
Conditions
Mutagenesis Chemotrophically grown R. sphaeroides were mutagenixed with nitrosoquanidine. Twenty milliliters of log-phase cells (5 x 108 cells/ml) was harvested by centrlfugation, washed, and resuspended in 5 ml of Tris-malate (T-M) buffer (0.2 M Trls. 0.2 M malate, pH 6.0) with 100 gg/ml of nitrosoguanidlne. The suspension was incubated for 20 min at 34°C. after which time the cells were harvested by centrifugation, washed twice in T-M buffer, and inoculated into 20 ml of PY medium. After overnight chemotrophic growth, the cells were harvested, washed twice with ice-cold SIS, and resuspended in 10 ml of SIS, and 0.5
* Abbrevations used PY, SIS, Sistrom’s minimal salts; kanamycin; Tc, tetracycline; by a grant from the streptomycin; Rif, rifampicin; ing. 229
and Pseudomonas
work was supported Science Foundation.
were routinely grown on liquid peptone-yeast extract (PYlz medium (3 g of peptone, 3 g of yeast extract/liter, pH 7.0) at 37 or 34”C, respectively. For plating, agar was added to the medium to a final concentration of 1.5% for bottom agar or 0.7% for soft agar. Rhndopseudomonas sphaeroides and Rhodopseudomonas capsulata were grown on Sistrom’n minimal salts @IS) medium (5) at 34°C. Phototrophic growth was accomplished by inoculating cells into a screwcapped tube completely filled with SIS and incubating at a light intensity of 700 fc. Phototrophic growth on liquid medium was accomplished by sealing plates in a brewer jar made anaerobic by a Gaspak (BBL, Cockeysville, Maryland) and incubating the jar at 34°C with 700 fc incident on the jar. For chemotrophic growth, cells were grown in Delong flasks filled to 20% capacity in a water bath with shaking. Media were supplemented with amino acids ‘(40 cg/ml, final concentration) when necessary to allow the growth of auxotrophs.
ooO3-9861/78/1871-0153~.00/0 Copyright 0 1978by Academic Press, I,,c. All rights of reproduction in any form reserved.
aeruginosa
peptone-yeast extract; T-M, Tris-malate; K, Cb, carbenicilhn; Sm, EOP, efficiency of plat-
MILLER
230
AND
KAPLAN
TABLE
I
BACTERIAL STRAINS USED IN THIS INWWTIGATION Strain
Plasmid
Genotypes
RP4 R6886 None PLM2
pro, met, F+ (K, Tc, Cb) thr, ku, thi, str hp (am), lac bm), [(K, Tc bm), Cb (am)1
Source
Escherichia coli 553 (RP4) 553.1 (R6886) 0500 CA274
pro, met, F+ (K, Tc, Cb)
R. W. Hedges R. W. Hedges L. Mindich
Pseudomonas aeruginosa PAL (R1822) PA025
R1822 R68.45
Prototroph (K, Tc, Cb) arg, leu (K, Tc, Cb)
R. H. Olsen J. D. Wall
Rhodopseudomonas sphaeroides 2.4.1 cu602 CU616 cm04
None None None RP4
Prototroph
trp, rib crt hp, his, rib str, crt Prototroph
(K, Tc, Cb)”
This study This study This study
Prototroph,
rif
H-C. Yen
Rhodopseudomonas capsulata SB1003 a The phenotype resistant (see text).
None
of CU604 is Cb sensitive,
although
ml was inoculated into SIS. After two doublings, sodium ampicillin was added to a final concentration of 100 ~/ml. When the optical density of the culture declined, the cells were harvested and allowed to recover in PY medium overnight. After three cycles of ampicillin enrichment, the cells were diluted and plated on SIS minimal agar supplemented with 2 ml of nutrient broth/liter. After 4 days of growth, minute colonies were picked to PY agar and tested for auxotrophy by replica plating to SIS minimal agar supplemented with amino acids.
Antibiotics All antibiotics were freshly prepared before use. With the exception of rifampicin, all were dissolved in distilled water and filter-sterilized. Rifampicin was dissolved in absolute methanol and used without further sterilization. Final concentrations of antibiotics used for E. coli and P. aeruginosa were: kanamycin (K), 100 &ml; tetracycline (Tc), 25 &ml, carbenicillin (Cb), 100 &ml. Final concentrations for R. sphaeroides and R. capsulata were: K, 20 pg/ml, Tc, 10 pg/ml; Cb, 50 pgg/ml; streptomycin (Sm) and rifampicin (Rif), 50 &ml.
Mating Techniques Liquid mating. Donor and recipient cells were grown either chemotrophically or phototrophically to 5 x l@ cells/ml, harvested, and washed 2~ in ice-cold SIS. The pellets were resuspended in SIS to a final concentration of 1 x 10’ cells/ml. One milliliter each of donor and recipient was added to a 13-mm test tube and incubated statically at 34’C for 4 h. The mixture was then diluted, and 0.1~ml aliquots of each dilution were mixed with 3.0 ml of SIS soft agar and plated on SIS agar at 34°C. After 18 h the plates were overlayed with 10 ml of SIS soft agar containing the selective
mating to E. coli indicates
that the genotype
is Cb
antibiotics to yield a final concentration in the plates as mentioned above. Plate mating. Plate mating differed from liquid mating only in that there was no 34°C incubation prior to dilution and plating.
Plasmid Isolation Plasmids were isolated from E. coli following the procedure of Meyers et al. (6). Plasmids were isolated from R. sphaeroides as follows: Cells were grown chemotrophically in 750 ml of SIS medium. The culture was sparged with a mixture of air and oxygen to ensure an oxygen content of 13 ppm. When cell density reached 1 x 10’ cells/ml, the culture was harvested by centrifugation and lysed using the method of Grinstead et al. (7) with the omission of the shearing step. The lysate was then centrifuged at 18,000 g (25 min, 2°C) and the resulting cleared lysate was subjected to the polyethylene glycol precipitation technique of Humphreys et al. (8). Agarose gel electrophoresis was carried out in the buffer system described by Greene et al. (9). 0.7% Agarose gels were cast as vertical slabs, 8 X 10 X 0.3 cm. After casting, a piece of silk was used across the bottom of the gel to prevent slippage during electrophoresis. A current of 60 mA was applied to the gels for 1 h or until the dye front was at the bottom of the gel. DNA was visualized by soaking the gel for 1 h in electrophoresis buffer containing 0.4 pg of ethidium bromide/ml and examining the gel under short-wave uv light. RESULTS
Intra- and Interspecies Transfer of Plasmid RP4 Initial experiments involving the transfer of RP4 from Escherichia coli to Rhodo-
PLASMIDS
231
IN Rhodopseudomonas sphaeroides
pseudomonas sphaeroides were performed using the liquid mating technique, followed by phototrophic growth on SIS minimal agar supplemented with kanamycin. These experiments resulted in a frequency of transfer of the selected marker of 1 x 10V6 per donor cell. Control plates showed no spontaneous resistance to kanamycin. were colonies Kanamycin-resistant picked and tested for resistance to the unselected markers Tc and Cb. All colonies tested were found to be resistant to Tc, but not Cb. These colonies were then streaked for isolation on PY agar containing K and Tc to ensure that all isolates were free of residual E. coli. After three streakings, one clone, designated CU604, was picked for further study. Since the mechanisms of action of K and Tc both involve inhibition of protein synthesis at the level of the ribosome, and since Cb resistance was not seen in any of the K, Tc-resistant cells, it was considered possible that resistance to CU604 was due to a single chromosomal mutation resulting in resistance to both drugs, rather than to the presence of the plasmid. In view of the fact that spontaneous resistance to kanamycin was not seen on control plates, the possibility was deemed unlikely, but further tests were performed to eliminate the possibility of a single-site mutation. Tests to determine the stability of the kanamycin and tetracycline resistance were performed by growing CU604 chemotrophically for a total of 20 generations in the absence of antibiotics. At the end of this time, viable counts of the culture were made on SIS, SIS + K, and SIS + Tc. The results of such testing showed that CU604 retained the kanamycin resistance marker in all cells, while the Tc marker was retained in less than 1% of the cells, a clear indication that the resistance to K was independent of Tc resistance. Subsequent tests on CU604 which had been maintained on K and Tc for 8 months indicated that both K and Tc resistances were extremely stable. To establish unequivocally that K and Tc resistance was the result of the presence of RP4, CU604 was mated to E. coli C600 and R. sphaeroides, CU602, and the result-
ing Tc-resistant transcipients were used for plasmid isolation. CU602 was used as a recipient in the intraspecies cross, since it is both Rif resistant (allowing counterselection against CU604) and green (allowing clear distinction of transcipients from the red donor). Both liquid and plate matings were performed for the intra- and interspecies crosses in an attempt to optimize mating conditions. As can be seen from Table II, the plate mating technique was more efficient as a condition for plasmid transfer, and was adopted for routine use. Additionally, Table II shows that, in the interspecies cross, all E. coli transcipients selected for Tc resistance also acquired resistance to Cb, while 78% acquired K resistance, indicating that the plasmid remains functionally intact in R. sphaeroides, although the p-lactamase activity responsible for Cb resistance apparently is not expressed or is ineffective in protecting R. sphaeroides. The fact that the plasmid remains intact is borne out by the fact that a plasmid could be isolated from both E. coli and R. sphaeroides transcipients which corn&rated on agarose gels with RP4 (Fig. 1). While R. sphaeroides 2.4.1 normally carries at least two (10) and possibly three covalently closed circular DNA species (ll), none of these con&rates with RP4. As can be seen in Fig. 1, R. sphaeroides 2.4.1 carries two plasmids whose molecular weights have been determined to be 7.5 and 2.8 x 107.E. coli C600 carries no plasmid. CU604 carries a third plasmid with a molecular weight of 4.0 X 10’ and whose presence confers kanamycin and tetracycline resistance. Kanamycin, tetracycline, and carbenicillin resistTABLE II CONDITIONS OF MATING Recipient”
cu602 cm02 C600 C600
Mating
Liquid Plats Liquid Plate
Selected marker transfer frequency per donor 1.4 x 9.8 x
