Vol. 130, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 529-531 Copyright © 1977 American Society for Microbiology
New R Plasmid-Mediated Restriction-Modification System of Deoxyribonucleic Acid Conferred by Group E R Plasmids TOSHIHIKO ARAI AND TAKASHI AOKI* Department of Microbiology, Keio University School of Medicine, Tokyo, 160 Japan, and Department of Fisheries, Faculty of Agriculture, Miyazaki University, Miyazaki, 880 Japan*
Received for publication 19 October 1976
A new R plasmid-mediated restriction-modification system of deoxyribonucleic acid was identified. This system is specific for group E plasmids which have been detected in unidentified marine Vibrio fish pathogens.
Some plasmids are already known to have tion patterns of phage X and Ti on various R+ restriction-modification systems for deoxyribo- strains of Escherichia coli K-12. Host strains nucleic acid (DNA) (5, 9, 10). That is, some for R plasmids were E. coli K-12: RC85nal for plasmids have genes for restriction enzymes phage Ti and C600 for phage XcI857. One-tenth which recognize in-coming foreign DNA in a milliliter of the diluted phage lysate was added cell and cut this DNA down to small fragments. to 0.1 ml of the indicator strain carrying an R Genetic materials in the cell are already modi- plasmid in 3 ml of L soft agar, poured onto an L fied by the coupled modification enzyme, so this agar plate (7), and incubated for overnight at DNA is prevented from digestion by the restric- 370C. Plaques appearing on each R+ indicator tion endonuclease. Thus, the cells that have a strain were counted, and the EOP was calcuspecific R plasmid are prevented from further lated as relative numbers of plaques on each genetic changes which otherwise may be trans- indicator strain. Single plaques on each indicamitted to them by foreign DNA. tor strain were picked and the phages were This self-preserving function which keeps propagated on the same indicator strain by the bacterial genetic constitutions unchanged is confluent lysis method. The phage lysate in soft rather essential for bacteria to keep their spe- agar was transferred into L broth and the agar cies specificity. In fact, most bacteria carry at was removed by centrifugation. Thus a stock least one specific restriction-modification sys- chloroformed lysate of each modified phage was tem (8). There is no necessity for plasmids to obtained and then the EOP was tested on varhave these systems. But, if plasmids them- ious indicator strains. These modified phages selves also come from a primitive bacterial plated on the R- strain were again purified on chromosome, it is no wonder that some of them the R- strain and their EOP was tested on the have specific systems. In this sense, plasmids same series of indicator strains. Twenty-six R plasmids detected in unidentithat have specific restriction-modification systems could be considered to be younger plas- fied marine Vibrio isolated from different farms mids in their evolutional histories, because the were transferred to E. coli RC85nal. Eighteen establishment of a specific restriction-modifica- out of 26 R plasmids gave the host bacteria the tion system could be understood as one of the ability to reduce the EOP of phage Ti. Phage signs of the establishment of the bacterial sub- Ti lysates grown on each of these R+ bacteria species. The fact that replicons of these plas- turned out to have the same EOP on all of these mids which have the same specificity always R+ strains as well as on the R- strain, and after belong to a single incompatibility group fits regrowth on the R- strain, it developed that they were again restricted by R+ bacteria. this idea well. The simple method to identify the specifici- Thus, it is concluded that these R plasmids ties is to estimate the efficiencies of plating detected in marine Vibrio have their specific restriction-modification system and that they (EOP) of temperate or weak virulent phages. We have isolated many R plasmids in uni- are all classified into the single group by the dentified Vibrio strains pathogenic for seawa- specificity for restriction-modification. Then we compared this system represented ter fish (3) and most of them turned out to have restriction systems (4). These R plasmids are by R plasmid pJA4620 with other previously all classified into a single group by their incom- reported R plasmid-mediated systems. The folpatibility (1). Therefore, we also examined lowing incompatibility groups were transferred their specificities by their restriction-modifica- to RC85nal and C600: FII R, 1RDK; FIV, R124; 529
530
J. BACTERIOL.
NOTES
N, N-3 and R245; and E, pJA4620. Phage Ti and XcI857 lysates grown on these strains as well as on the R- strain were examined for their EOP on these strains. Results for phages Ti and XcI857 are shown in Tables 1 and 2, respectively. Both results clearly indicate that the restriction-modification system specific for group E plasmids is different from three other plasmid-mediated systems. Group E plasmids have been detected only from pathogenic fish Vibrio such as Vibrio anguillarum and unidentified marine Vibrio (1). Since Vibrio spp. are salt dependent and quite different in their physiolocp al characters from other bacteria living in freshwater environments, including Enterobacteriaceae, it is no wonder that R plasmids in this specie-s are quite different in their character from other plasmids in bacteria living in freshwater environments. Although group E plasmids have been detected only in pathogenic fish Vibrio, fish diseases caused by Vibrio spp. occur in both freshwater and saltwater fish. From freshwater fish, we have mainly isolated V. anguillarum, and, from seawater fish, we have isolated unidentified marine Vibrio (2, 3). R plasmids detected in V. anguillarum were examined for their ability to restrict phage DNA. None out of 58 R plasmids conferred to their host strains the functions of restriction and modification of DNA. Therefore, it is suggested that this restriction-modification system of DNA is specific only for R plasmids detected in seawater Vibrio.
As far as we have examined, all the group N and FIV R plasmids have group-specific restriction-modification systems. Thus, R plasmidmediated restriction-modification systems are useful for identification of specific R plasmids as well as the tools for gene manipulations (6). We were informed by H. W. Boyer of University of California, San Francisco (personal communiication), that he did not detect any endonucleolytic activity in the cell-free extract of the cells carrying pJA4620 by the EcoRI and EcoRII in vitro assay system (8). This suggests that this group E-specific system requires something extra for its activity. This research was partly supported by grants-in-aid from the Ministry of Education and Science, Japan. LITERATURE CITED 1. Aoki, T., T. Arai, and S. Egusa. 1975. R factors detected
2.
3. 4.
5.
from Vibrio anguillarum and marine Vibrio, p. 223226. In S. Mitsuhashi and H. Hashimoto (ed.), Microbial drug resistance. University of Tokyo Press, Tokyo. Aoki, T., S. Egusa, and T. Arai. 1974. Detection of R factors in naturally occurring Vibrio anguillarum strains. Antimicrob. Agents Chemother. 6:534-538. Aoki, T., S. Egusa, and T. Watanabe. 1973. Detection of RI bacteria in cultured marine fish, Yellowtails (Seriola Quinqueradiata). Jpn. J. Microbiol. 17:7-12. Aoki, T., S. Egusa, T. Watanabe, K. Kitai, and K. Akagawa. 1973. Some characters of R factors detected in Vibrio spp. isolated from Yellowtail (Seriola quinqueradiata). Jpn. J. Bacteriol. (in Japanese) 28:288. Bannister, D., and S. W. Glover. 1968. Restriction and modification of bacteriophages by RI strains ofEsche-
TABLE 1. EOP of phage Ti on E. coli K-12:RC85nal carrying various R plasmids EOP of phage Ti grown on RC85nal carrying R plasmid:
Indicator strain RC85nal
RR124 R245 N-3 1RDK
pJA4620
R-
1 1.0 x 10-3 1.8 x 10-4 2.2 x 10-4 1.7 x 10-2 1.7 x 10-2
R124 (FIV) 1
2.6 2.7 1.5 1.8
1 x 10-4 x 10-4 x 10-2 x 10-2
R245 (N) 1
N-3 (N) 1
1RDK (FII) 1
8.6 x 10-4 1 1 1.5 x 10-2 2.2 x 10-2
9.6 x 10-4 1 1 1.6 x 10-2 2.6 x 10-2
1.1 X 10-3 4.0 x 10-4 5.1 x 10-4 1 3.0 x 10-2
pJA4620 (E) 1 1.0 X 10-3 3.5 x 10-4 4.1 x 10-4 1.7 x 10-2 1
TABLE 2. EOP of phage XcI857 on E. coli K-12:C600 carrying various R plasmids Indicator strain C600
RR124
R245 N-3 1RDK pJA4620
EOP of phage XcI857 grown on C600 carrying R plas; iid: R124 (FIV) R245 (N) N-3 (N) 1RDK (FII)
R
1 1
1
3.2 6.4 5.0 3.9 9.8
x x x x x
10-4 10-2
10-2 10-4 10-3
2.6 4.2 2.3 2.3
x x x x
10-2 10-2 10-4 10-3
1 1.8 x 10-4 1 1 3.5 x 10-4 1.7 x 10-3
1
1.1
X
10-4
1 1 4.1 x 10-4 3.3 x 10-3
1 2.1 x 10-4 6.0 x 10-2 6.0 x 10-2 1 9.4 x 10-3
pJA4620 (E)
1 1.9 X 10-4 2.1 x 10-2 2.6 x 10-2 2.4 x 10-4 1
VOL. 130, 1977 richia coli K-12. Biochem. Biophys. Res. Commun. 30:735-738. 6. Cohen, S. N. 1975. Manipulation of genes. Sci. Am. 233:24-33. 7. Lennox, E. S. 1955. Transduction of linked genetic characters of host by bacteriophage P1. Virology 1:190-206. 8. Meselson, M., R. Yuan, and J. Heywood. 1972. Restriction and modification of DNA. Annu. Rev. Biochem.
NOTES
531
41:447-466. 9. Watanabe, T., H. Nishida, C. Ogata, T. Arai, and S. Sato. 1964. Episome-mediated transfer of drug resistance in Enterobacteriaceae. VII. Two types of naturally occurring R factors. J. Bacteriol. 88:716-726. 10. Yoshimori, R., D. Roulland-Dussoix, and H. W. Boyer. 1972. R factor-controlled restriction and modification of deoxyribonucleic acid: restriction mutants. J. Bacteriol. 112:1275-1279.
JOURNAL OF BACTERIOLOGY, Apr. 1977, p. 529-531 Copyright © 1977 American Society for Microbiology
New R Plasmid-Mediated Restriction-Modification System of Deoxyribonucleic Acid Conferred by Group E R Plasmids TOSHIHIKO ARAI AND TAKASHI AOKI* Department of Microbiology, Keio University School of Medicine, Tokyo, 160 Japan, and Department of Fisheries, Faculty of Agriculture, Miyazaki University, Miyazaki, 880 Japan*
Received for publication 19 October 1976
A new R plasmid-mediated restriction-modification system of deoxyribonucleic acid was identified. This system is specific for group E plasmids which have been detected in unidentified marine Vibrio fish pathogens.
Some plasmids are already known to have tion patterns of phage X and Ti on various R+ restriction-modification systems for deoxyribo- strains of Escherichia coli K-12. Host strains nucleic acid (DNA) (5, 9, 10). That is, some for R plasmids were E. coli K-12: RC85nal for plasmids have genes for restriction enzymes phage Ti and C600 for phage XcI857. One-tenth which recognize in-coming foreign DNA in a milliliter of the diluted phage lysate was added cell and cut this DNA down to small fragments. to 0.1 ml of the indicator strain carrying an R Genetic materials in the cell are already modi- plasmid in 3 ml of L soft agar, poured onto an L fied by the coupled modification enzyme, so this agar plate (7), and incubated for overnight at DNA is prevented from digestion by the restric- 370C. Plaques appearing on each R+ indicator tion endonuclease. Thus, the cells that have a strain were counted, and the EOP was calcuspecific R plasmid are prevented from further lated as relative numbers of plaques on each genetic changes which otherwise may be trans- indicator strain. Single plaques on each indicamitted to them by foreign DNA. tor strain were picked and the phages were This self-preserving function which keeps propagated on the same indicator strain by the bacterial genetic constitutions unchanged is confluent lysis method. The phage lysate in soft rather essential for bacteria to keep their spe- agar was transferred into L broth and the agar cies specificity. In fact, most bacteria carry at was removed by centrifugation. Thus a stock least one specific restriction-modification sys- chloroformed lysate of each modified phage was tem (8). There is no necessity for plasmids to obtained and then the EOP was tested on varhave these systems. But, if plasmids them- ious indicator strains. These modified phages selves also come from a primitive bacterial plated on the R- strain were again purified on chromosome, it is no wonder that some of them the R- strain and their EOP was tested on the have specific systems. In this sense, plasmids same series of indicator strains. Twenty-six R plasmids detected in unidentithat have specific restriction-modification systems could be considered to be younger plas- fied marine Vibrio isolated from different farms mids in their evolutional histories, because the were transferred to E. coli RC85nal. Eighteen establishment of a specific restriction-modifica- out of 26 R plasmids gave the host bacteria the tion system could be understood as one of the ability to reduce the EOP of phage Ti. Phage signs of the establishment of the bacterial sub- Ti lysates grown on each of these R+ bacteria species. The fact that replicons of these plas- turned out to have the same EOP on all of these mids which have the same specificity always R+ strains as well as on the R- strain, and after belong to a single incompatibility group fits regrowth on the R- strain, it developed that they were again restricted by R+ bacteria. this idea well. The simple method to identify the specifici- Thus, it is concluded that these R plasmids ties is to estimate the efficiencies of plating detected in marine Vibrio have their specific restriction-modification system and that they (EOP) of temperate or weak virulent phages. We have isolated many R plasmids in uni- are all classified into the single group by the dentified Vibrio strains pathogenic for seawa- specificity for restriction-modification. Then we compared this system represented ter fish (3) and most of them turned out to have restriction systems (4). These R plasmids are by R plasmid pJA4620 with other previously all classified into a single group by their incom- reported R plasmid-mediated systems. The folpatibility (1). Therefore, we also examined lowing incompatibility groups were transferred their specificities by their restriction-modifica- to RC85nal and C600: FII R, 1RDK; FIV, R124; 529
530
J. BACTERIOL.
NOTES
N, N-3 and R245; and E, pJA4620. Phage Ti and XcI857 lysates grown on these strains as well as on the R- strain were examined for their EOP on these strains. Results for phages Ti and XcI857 are shown in Tables 1 and 2, respectively. Both results clearly indicate that the restriction-modification system specific for group E plasmids is different from three other plasmid-mediated systems. Group E plasmids have been detected only from pathogenic fish Vibrio such as Vibrio anguillarum and unidentified marine Vibrio (1). Since Vibrio spp. are salt dependent and quite different in their physiolocp al characters from other bacteria living in freshwater environments, including Enterobacteriaceae, it is no wonder that R plasmids in this specie-s are quite different in their character from other plasmids in bacteria living in freshwater environments. Although group E plasmids have been detected only in pathogenic fish Vibrio, fish diseases caused by Vibrio spp. occur in both freshwater and saltwater fish. From freshwater fish, we have mainly isolated V. anguillarum, and, from seawater fish, we have isolated unidentified marine Vibrio (2, 3). R plasmids detected in V. anguillarum were examined for their ability to restrict phage DNA. None out of 58 R plasmids conferred to their host strains the functions of restriction and modification of DNA. Therefore, it is suggested that this restriction-modification system of DNA is specific only for R plasmids detected in seawater Vibrio.
As far as we have examined, all the group N and FIV R plasmids have group-specific restriction-modification systems. Thus, R plasmidmediated restriction-modification systems are useful for identification of specific R plasmids as well as the tools for gene manipulations (6). We were informed by H. W. Boyer of University of California, San Francisco (personal communiication), that he did not detect any endonucleolytic activity in the cell-free extract of the cells carrying pJA4620 by the EcoRI and EcoRII in vitro assay system (8). This suggests that this group E-specific system requires something extra for its activity. This research was partly supported by grants-in-aid from the Ministry of Education and Science, Japan. LITERATURE CITED 1. Aoki, T., T. Arai, and S. Egusa. 1975. R factors detected
2.
3. 4.
5.
from Vibrio anguillarum and marine Vibrio, p. 223226. In S. Mitsuhashi and H. Hashimoto (ed.), Microbial drug resistance. University of Tokyo Press, Tokyo. Aoki, T., S. Egusa, and T. Arai. 1974. Detection of R factors in naturally occurring Vibrio anguillarum strains. Antimicrob. Agents Chemother. 6:534-538. Aoki, T., S. Egusa, and T. Watanabe. 1973. Detection of RI bacteria in cultured marine fish, Yellowtails (Seriola Quinqueradiata). Jpn. J. Microbiol. 17:7-12. Aoki, T., S. Egusa, T. Watanabe, K. Kitai, and K. Akagawa. 1973. Some characters of R factors detected in Vibrio spp. isolated from Yellowtail (Seriola quinqueradiata). Jpn. J. Bacteriol. (in Japanese) 28:288. Bannister, D., and S. W. Glover. 1968. Restriction and modification of bacteriophages by RI strains ofEsche-
TABLE 1. EOP of phage Ti on E. coli K-12:RC85nal carrying various R plasmids EOP of phage Ti grown on RC85nal carrying R plasmid:
Indicator strain RC85nal
RR124 R245 N-3 1RDK
pJA4620
R-
1 1.0 x 10-3 1.8 x 10-4 2.2 x 10-4 1.7 x 10-2 1.7 x 10-2
R124 (FIV) 1
2.6 2.7 1.5 1.8
1 x 10-4 x 10-4 x 10-2 x 10-2
R245 (N) 1
N-3 (N) 1
1RDK (FII) 1
8.6 x 10-4 1 1 1.5 x 10-2 2.2 x 10-2
9.6 x 10-4 1 1 1.6 x 10-2 2.6 x 10-2
1.1 X 10-3 4.0 x 10-4 5.1 x 10-4 1 3.0 x 10-2
pJA4620 (E) 1 1.0 X 10-3 3.5 x 10-4 4.1 x 10-4 1.7 x 10-2 1
TABLE 2. EOP of phage XcI857 on E. coli K-12:C600 carrying various R plasmids Indicator strain C600
RR124
R245 N-3 1RDK pJA4620
EOP of phage XcI857 grown on C600 carrying R plas; iid: R124 (FIV) R245 (N) N-3 (N) 1RDK (FII)
R
1 1
1
3.2 6.4 5.0 3.9 9.8
x x x x x
10-4 10-2
10-2 10-4 10-3
2.6 4.2 2.3 2.3
x x x x
10-2 10-2 10-4 10-3
1 1.8 x 10-4 1 1 3.5 x 10-4 1.7 x 10-3
1
1.1
X
10-4
1 1 4.1 x 10-4 3.3 x 10-3
1 2.1 x 10-4 6.0 x 10-2 6.0 x 10-2 1 9.4 x 10-3
pJA4620 (E)
1 1.9 X 10-4 2.1 x 10-2 2.6 x 10-2 2.4 x 10-4 1
VOL. 130, 1977 richia coli K-12. Biochem. Biophys. Res. Commun. 30:735-738. 6. Cohen, S. N. 1975. Manipulation of genes. Sci. Am. 233:24-33. 7. Lennox, E. S. 1955. Transduction of linked genetic characters of host by bacteriophage P1. Virology 1:190-206. 8. Meselson, M., R. Yuan, and J. Heywood. 1972. Restriction and modification of DNA. Annu. Rev. Biochem.
NOTES
531
41:447-466. 9. Watanabe, T., H. Nishida, C. Ogata, T. Arai, and S. Sato. 1964. Episome-mediated transfer of drug resistance in Enterobacteriaceae. VII. Two types of naturally occurring R factors. J. Bacteriol. 88:716-726. 10. Yoshimori, R., D. Roulland-Dussoix, and H. W. Boyer. 1972. R factor-controlled restriction and modification of deoxyribonucleic acid: restriction mutants. J. Bacteriol. 112:1275-1279.
