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Stabilization of the cloning vector pACYC184 by insertion of F plasmid leading region sequences
PLASMID
11,
272-275 (1984)
Stabilization of the Cloning Vector pACYC184 by Insertion of F Plasmid Leading Region Sequences ANIMESH
RAY’
AND RON SKURRAY’
Department of Microbiology, Monash University, Clayton, Victoria, Australia 3168 Received Nov. 9, 1983; revised February 28, 1984 The leading region of the F plasmid is, by definition, the first part of the plasmid DNA to be transferred to the recipient cell during conjugation. Restriction fragments of the leading region, when cloned into the plasmid vector pACYC184, extended the maintenance of the normally unstable p 1SAderived vector replicon in ret+ Escherichia coli K- 12 cells. Mutations in the host’s general recombination systemswere found to influence the maintenance of thesehybrid plasmids.
The genes involved in conjugational DNA transfer map between 66.7F and 1OOFon the lOO-kb genetic map of the F plasmid (I); the primary replication-incompatibility region, when projected on this map, is located between 48.3 and 53.8F (2). Sandwiched between the primary replication-incompatibility region and the transfer (@a) genes, a 12.9-kb segment of F DNA from the EcoRI site at 53.8F to the origin of transfer oriT at 66.7F (Fig. l), earlier defined as the leading region in F DNA transfer (3), has been shown to express at least five polypeptides in maxicells (4). We have now addressed the question of possible functions associated with this segment of F DNA. On the basis of our observations that cloned segments of the leading region in the multicopy vector pACYC 184 (5) did not show incompatibility towards an incoming F’luc+ plasmid JCFLO (6), and that these hybrid plasmids were unable to replicate in a thermosensitive DNA polymerase I mutant strain (data not shown), it was concluded that the leading region of the F plasmid does not code either for additional IncFI incompatibility or for a DNA polymerase I-independent replicon. In addition to an essential replication system, stable maintenance of a plasmid in a bacterial population requires a mechanism for effective partitioning; plasmids such as Xdv, ’ Present address:Institute of Molecular Biology, University of Oregon, Eugene, Oreg. 97403. * To whom reprint requests should be addressed. 0147-619X/84 $3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
and in vitro constructed multicopy plasmids such as pBR322, lack a partitioning mechanism (Par-) and are lost from the population at a rapid rate (7-9). The plasmid vector pACYC 184 has also been shown to be unstable (IO) and this instability is believed to be due to loss of the p 15A partitioning (par) locus during its construction (5,10). Since the leading region is physically adjacent to the region of F replication and partitioning, we have investigated whether any part of the leading region could stabilize the Par- pl5A replicon of the vector pACYC 184. Nearly 100% of cells in a population of strain ABl157 (1 I) retained the hybrid plasmid pRS2062 (53.8-64.7F EcoRI fragment f3 of F cloned into pACYCl84, Fig. 1) for at least 50 generations of nonselective growth whereas the vector pACYC 184 was lost rapidly after a lag of 20 generations (Fig. 2A). The same EcoRI fragment inserted into pACYC184 in the opposite orientation, as in pRS2059 (4), showed an identical effect on the maintenance of the Par- pl5A replicon by AB 1157 (data not shown). The same extent of stability was observed (Fig. 2A) for the plasmid pRS1708 (56.4-67.lF Sal1 fragment cloned into pACYCl84, Fig. 1). Since only the 56.4-64.7F segment of the leading region is common to these plasmids, we suggest that the sequence of F DNA responsible for stable maintenance of the pACYC 184 replicon in AB1157 cells lies within 56.4-64.7F. The increased stability of pRSl708 and 272
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273
.f5-+-f3--f6-
FIG. I. Summary of F-derived recombinant plasmids used. pRS2062 has been described previously (4), and pRS 1708and pRS 1705are described in the accompanying article (20); the vector for all three plasmids was pACYCl84. The coordinates, based on a lOO-kb F map (I), of relevant restriction sites are indicated on EcoRl fragments f5, f3, and f6 of F, the enzyme abbreviations are: B, BarnHI; Bg, BglII; E, EcoRI; S, &/I. The locations of the origins of replication ori-l and ori- (2), the primary replication region Prep (2), b-encoded partition region par (26,27), incll incompatibility (28), x$(18), and the transfer genestrukf, J. Y, A, L, E, K, B, P (I) are indicated on the physical map; the arrowhead at oriT (66.7F) indicates the direction of conjugational DNA transfer (I).
pRS2062 compared to pACYCl84 in strain copy numbers of these plasmids compared to AB1157 could be due either to efficient par- the vector’s. In order to examine the second titioning of the hybrid plasmids or to increased possibility, plasmid copy numbers were measured by the dye-buoyant isopycnic centrifugation method using [3H]thymidine pulselabeled DNA (12). The estimated copy numbers of pACYC 184, pRS 1708, and pRS2062 were 30, 8.5, and 6.4, respectively, in ABI 157 cells; a copy number of 18 for pACYCl84 has been previously reported (5). While these results suggest that the increased stability of pRS1708 and pRS2062 cannot be due to higher plasmid copy numbers relative to that of the vector, it should be noted that the method employed would not detect increased copy numbers of linear or nonclosed circular FIG. 2. Stability of plasmids carrying segments of the molecules. leading region. Strains containing the plasmids were plated In the light of the observations that host on selective L agar, single colonies suspended in nonserecombination systems are involved in the lective L broth (4) and immediately diluted in fresh L broth to lo4 cells/ml and incubated at 37°C. These cul- maintenance of ColEI (13) and CloDF 13 (14) tures, after appropriate dilution, were further subcultured plasmids, it was thought appropriate to study at 12-hintervals and dilutions were plated on nonselective the effectsof specific recombination mutations L agar plates for viable counting. After overnight incubation of the plates at 37”C, the plates with 100-500 on maintenance of pRS1708 and pRS2062. colonies were replica plated on suitable selective media The rates of loss of pACYCl84 from poputo determine the proportion of antibiotic resistant cells. lations of the recA-I3 strain AB2463 (1 Z), and Strains carrying pRS1708 (0) were resistant to chlor- the r-e&-21, recC-22 strain JC5519 (15), did amphenicol (25 pg ml-‘), strains carrying pRSl705 (m) not differ from that observedwith the isogenic and pRS2062 (V) were resistant to tetracycline (10 pg ml-‘), and those carrying pACYCl84 (0) were resistant ret+ strain (Fig. 2). On the other hand, to both antibiotics. Generation time was computed from pRS2062 was lost at a very rapid rate from the viable counts and 12-hcorrespondedto approximately cultures of each of the two recombination10 doublings for each of the strains used. Stability of the deficient strains (Figs. 2B and C). In recA muplasmids were determined in the ret+ strain ABl157 (A) tant cells, both the RecF and RecBC pathways and its isogenic derivatives, the recA-13 strain AB2463 (B), and the recE-21, recC-22 strain JC5519 (C). Each of recombination are inoperative, whereas in point denotesan averageof at least two independent mea- a recBC mutant, the RecBC pathway is surements. blocked but the RecF pathway remains intact
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(26). On this basis,our data may be interpreted to indicate that stabilization of the Par- pl5A replicon by the 56.4-64.7F segment of the leading region is mediated via the RecBC pathway of recombination which requires both the recA and recBC gene products. However, the pleiotropic nature of the RecA protein leaves open the possibility of other interpretations. Maintenance of pRS 1708, was not influenced by the recA mutation (Fig. 2B) and only a small, but reproducible, instability was apparent in the recBC mutant strain (Fig. 2C). These data, taken together with the results above, would suggestthat the presence of the 64.7-66.7F segmentoverrides the requirement of at least the RecA protein for stabilization of the pACYCl84 replicon by the 56.4-64.7F segment of the leading region. Alternatively, the absence of the 53.8-56.4F region from pRS1708 may bypass this requirement. Further evidence against the involvement of plasmid copy number in the stabilization of these recombinant plasmids is provided by our observation that the copy numbers of most of them did not differ significantly in the recA mutant strain from those in the ret+ strain. pACYC 184 was present at 22 copies per genome equivalent in each of the two recombination-deficient strains, while pRS 1708 had 7.8 and 3.1 copies in the recA and recBC strains, respectively, compared to 8.5 copies in the ret+ strain. pRS2062 had a copy number of 7.3 in the recA strain but 14 in the recBC strain. Thus, the twofold increase in the copy number of pRS2062 did not lead to a stable inheritance of the plasmid by the recBC mutant strain, nor was the instability of pRS2062 in the recA strain due to a decreased copy number. However, the approximately threefold reduction in the copy number of pRS 1708 in the recBC mutant could explain the reduced stability exhibited by pRS1708 in that strain (Fig. 2C). In order to examine the effect of the 64.766.7F segment on maintenance of the Parpl5A replicon in the absence of the 56.464.7F segment, the segregation kinetics of pRS1705 (64.7-73.0F EcoRI fragment f6 of F cloned into pACYC 184, Fig. 1) were measured in all three strains (Fig. 2). The results,
while clearly indicating that this segment of the leading region is not sufficient by itself to bring about the maintenance function, do not exclude the possibility that this region overrides the RecA dependenceof the f3-mediated Par function as suggestedabove. In this connection, it should be emphasized that the stability effect discussed in this communication is not related to the orientation-dependent instability of oriT+ clones in F+ cells asdescribed by Everett and Willetts (17). First, our experiments were performed in F cells and secondly, the difference in the stability of pRSl705 and pRSl708 could not be due to an orientation-dependent oriT effect; oriT is inserted in the same relative orientation with respectto the direction of replication from the p15A origin in both plasmids. The leading region segment between 56.4F and 66.7F codes for at least five polypeptides of molecular weights 33,400, 27,800, 23,100, 14,400, and 11,000 (4). A protein similar in size and map location to the 23,100-Da polypeptide has recently been shown (18) to be a single-stranded DNA (ssDNA) binding protein, the product of the ssfgene located at the 59.3F BumHI site on the leading region (Fig. 1). The 33,400-Da polypeptide, which is considered to be the 6d protein described earlier (19) and a 13,500-Da protein which was tentatively identified before (4), have now been mapped between 64.7F and 66.6F although their promoter is thought to be located on f3 (20). Functions of these proteins, and of the three remaining polypeptides, are not known. In support of the role of a f3-encoded protein in the stabilization of pACYC 184, preliminary experiments have shown that pACYC 184 was retained at nearly 100% level over 60 generations of nonselective growth by AB 1157 cells which additionally carried EcoRI f3 cloned in the compatible vector RSF2 124 (21) but not by ABI 157 which carried the vector RSF2124 alone. Such a result indicates that the D-mediated Par function acts in trans. The E. coli ssDNA binding protein (a product of the chromosomally encoded ssb gene), besides being essential for E. co/i DNA replication (22) is known to modulate the action of several enzymes such as the RecA protein
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(23) and the RecBC enzyme (exonuclease V) (24). Similarity in the nucleotide sequences of the s.sband the F-specified &genes has prompted the speculation that the properties of the proteins they encode may also be similar (25). On this basis, and in the light of our observation that f3-specified maintenance function is dependent on RecA and RecBC proteins in the absenceof the 64.7-66.6F segment, it should be interesting to investigate the role of the ssf and ssb genes in plasmid maintenance. It is, of course, potentially possible that any of the remaining three polypeptides of f3 are involved and we are also testing the possibility that a polypeptide coded by the 64.7-66.6F segment rescuesthe RecA dependence of the maintenance function. In this communication we have described the maintenance of the normally unstable pACYC184 replicon by the leading region of F but have not attempted to determine whether this activity plays a role in the stable maintenance of the F plasmid itself. While a par region involved in the partitioning and maintenance of F has been mapped (26,27) within the primary F replication-incompatibility region (Fig. l), the leading region may well provide a secondary F Par function. It may, on the other hand, be involved in posttransfer repliconation/maintenance of the F DNA in the recipient cell. ACKNOWLEDGMENTS We thank Louise O’Gorman and Robert Cross for technical assistance, David Cram for useful discussions, Barbara Bachmann (E. coli Genetic Stock Center, Yale University School of Medicine) for strains carrying ret mutations, and K. Brooks Low for communicating results prior to publication. This work was supported, in part, by a grant from the Australian Research Grants Scheme; A.R. was supported by a Monash Postgraduate Scholarship.
REFERENCES 1. WILLEITS, N., AND SKURRAY, R., Annu. Rev. Genet. 14,41-76 (1980). 2. KOMAI, N., NISHIZAWA, T., HAYAKAWA, Y., MuROT%, T., AND MAT~UBARA, K., Mol. Gen. Genet. 186, 193-203 (1982).
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3. CRAM, D. S., DIBERARDINO, D., AND SKURRAY, R. A., Mol. Gen. Genet. 185, 186-188 (1982). 4. RAY, A., AND SKURRAY, R., Plasmid 9, 262-272 (1983). 5. CHANG, A. C. Y., AND COHEN, S. N., J. Bacterial. 134, 1141-l 156 (1978). ACHTMAN, M., WILLETTS, N., AND CLARK, A. J., J. Bacterial. 106, 529-583 (197 1). KOLTER, R., AND HELINSKI, D. R., Annu. Rev. Genet. 13,355-391 (1979). IMANAKA, T., AND AIBA, S., Ann. N. Y. Acad. Sci. 369, l-14 (1981). THOMPSON, R., In “Genetic Engineering” (R. Williamson, ed.), Vol. 3, pp. 2-41. Academic Press, New York, 1982. 10. MEACOCK, P. A., AND COHEN, S. N., Cell 20, 529542 (1980). 11. HOWARD-FLANDERS, P., AND THERIOT, L., Genetics 53, 1137-l 150 (1966). 12. OGURA, T., MIKI, T., AND HIRAGA, S., Proc. Natl. Acad. Ski. USA 11, 3993-3997 (1980). 13. REAM, L. W., CRISONA, N. J., AND CLARK, A. J., In “Microbiology1978” (D. Schlessinger, ed.), pp. 78-80. Amer. Sot. Microbial., Washington, D. C., 1978. 14. HAKKAART, M. J. J., VELTKAMP, E., AND NIJKAMP, H. J. J., Mol. Gen. Genet. 188, 338-344 (1982). 15. WILLETTS, N. S., AND CLARK, A. J., J. Bacterial. 100,231-239 (1969). 16. CLARK, A. J., Genetics 78, 259-271 (1974). 17. EVERETT, R., AND WILLETTS, N., EMBO J. 1,747753(1982). 18. KOLODKIN, A. L., CAPAGE, M. A., GOLUB, E. I., AND LOW, K. B., Proc. Natl. Acad. Sci. USA 80,44224426 (1983). 19. THOMPSON, R., AND ACHTMAN, M., Mol. Gen. Genet. 169,49-57 (1979). 20. CRAM, D., RAY, A., O’GORMAN, L., AND SKURRAY, R., Plasmid 11, 221-233 (1984). 21. SKURRAY, R. A., CLARK, A. J., UHLIN, B. E., NAGAISHI, H., AND HUA, S., In “Microbiology1978” (D. Schlessinger, ed.), pp. 192-196. Amer. Sot. Microbial., Washington, D. C., 1978. 22. MEYER, R. R., GLASSBERG,T., AND KORNBERG, A., Proc. Natl. Acad. Sci. USA 76, 1702-1705 (1979). 23. COHEN, S. P., RESNICK, J., AND SUSSMAN, R., J. Mol. Biol. 167, 901-909 (1983). 24. MACKAY, V., AND LINN, S., J. Biol. Chem. 251,37 163719 (1976). 25. CHASE, J. W., MERRILL, B. M., AND WILLIAMS, K. R., Proc. Natl. dead. Sci. USA 80, 5480-5484 ( 1983). 26. OGURA, T., AND HIRAGA, S., Cell3&35 l-360 (1983). 27. AUSTIN, S., AND WIERZBICKI, A., Plasmid 10, 7381 (1983). 28. GARDNER, R. C., MALCOLM, L., BERGQUIST, P. L., AND LANE, H. E. D., Mol. Gen. Genet. 188,345352 (1982).
11,
272-275 (1984)
Stabilization of the Cloning Vector pACYC184 by Insertion of F Plasmid Leading Region Sequences ANIMESH
RAY’
AND RON SKURRAY’
Department of Microbiology, Monash University, Clayton, Victoria, Australia 3168 Received Nov. 9, 1983; revised February 28, 1984 The leading region of the F plasmid is, by definition, the first part of the plasmid DNA to be transferred to the recipient cell during conjugation. Restriction fragments of the leading region, when cloned into the plasmid vector pACYC184, extended the maintenance of the normally unstable p 1SAderived vector replicon in ret+ Escherichia coli K- 12 cells. Mutations in the host’s general recombination systemswere found to influence the maintenance of thesehybrid plasmids.
The genes involved in conjugational DNA transfer map between 66.7F and 1OOFon the lOO-kb genetic map of the F plasmid (I); the primary replication-incompatibility region, when projected on this map, is located between 48.3 and 53.8F (2). Sandwiched between the primary replication-incompatibility region and the transfer (@a) genes, a 12.9-kb segment of F DNA from the EcoRI site at 53.8F to the origin of transfer oriT at 66.7F (Fig. l), earlier defined as the leading region in F DNA transfer (3), has been shown to express at least five polypeptides in maxicells (4). We have now addressed the question of possible functions associated with this segment of F DNA. On the basis of our observations that cloned segments of the leading region in the multicopy vector pACYC 184 (5) did not show incompatibility towards an incoming F’luc+ plasmid JCFLO (6), and that these hybrid plasmids were unable to replicate in a thermosensitive DNA polymerase I mutant strain (data not shown), it was concluded that the leading region of the F plasmid does not code either for additional IncFI incompatibility or for a DNA polymerase I-independent replicon. In addition to an essential replication system, stable maintenance of a plasmid in a bacterial population requires a mechanism for effective partitioning; plasmids such as Xdv, ’ Present address:Institute of Molecular Biology, University of Oregon, Eugene, Oreg. 97403. * To whom reprint requests should be addressed. 0147-619X/84 $3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
and in vitro constructed multicopy plasmids such as pBR322, lack a partitioning mechanism (Par-) and are lost from the population at a rapid rate (7-9). The plasmid vector pACYC 184 has also been shown to be unstable (IO) and this instability is believed to be due to loss of the p 15A partitioning (par) locus during its construction (5,10). Since the leading region is physically adjacent to the region of F replication and partitioning, we have investigated whether any part of the leading region could stabilize the Par- pl5A replicon of the vector pACYC 184. Nearly 100% of cells in a population of strain ABl157 (1 I) retained the hybrid plasmid pRS2062 (53.8-64.7F EcoRI fragment f3 of F cloned into pACYCl84, Fig. 1) for at least 50 generations of nonselective growth whereas the vector pACYC 184 was lost rapidly after a lag of 20 generations (Fig. 2A). The same EcoRI fragment inserted into pACYC184 in the opposite orientation, as in pRS2059 (4), showed an identical effect on the maintenance of the Par- pl5A replicon by AB 1157 (data not shown). The same extent of stability was observed (Fig. 2A) for the plasmid pRS1708 (56.4-67.lF Sal1 fragment cloned into pACYCl84, Fig. 1). Since only the 56.4-64.7F segment of the leading region is common to these plasmids, we suggest that the sequence of F DNA responsible for stable maintenance of the pACYC 184 replicon in AB1157 cells lies within 56.4-64.7F. The increased stability of pRSl708 and 272
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273
.f5-+-f3--f6-
FIG. I. Summary of F-derived recombinant plasmids used. pRS2062 has been described previously (4), and pRS 1708and pRS 1705are described in the accompanying article (20); the vector for all three plasmids was pACYCl84. The coordinates, based on a lOO-kb F map (I), of relevant restriction sites are indicated on EcoRl fragments f5, f3, and f6 of F, the enzyme abbreviations are: B, BarnHI; Bg, BglII; E, EcoRI; S, &/I. The locations of the origins of replication ori-l and ori- (2), the primary replication region Prep (2), b-encoded partition region par (26,27), incll incompatibility (28), x$(18), and the transfer genestrukf, J. Y, A, L, E, K, B, P (I) are indicated on the physical map; the arrowhead at oriT (66.7F) indicates the direction of conjugational DNA transfer (I).
pRS2062 compared to pACYCl84 in strain copy numbers of these plasmids compared to AB1157 could be due either to efficient par- the vector’s. In order to examine the second titioning of the hybrid plasmids or to increased possibility, plasmid copy numbers were measured by the dye-buoyant isopycnic centrifugation method using [3H]thymidine pulselabeled DNA (12). The estimated copy numbers of pACYC 184, pRS 1708, and pRS2062 were 30, 8.5, and 6.4, respectively, in ABI 157 cells; a copy number of 18 for pACYCl84 has been previously reported (5). While these results suggest that the increased stability of pRS1708 and pRS2062 cannot be due to higher plasmid copy numbers relative to that of the vector, it should be noted that the method employed would not detect increased copy numbers of linear or nonclosed circular FIG. 2. Stability of plasmids carrying segments of the molecules. leading region. Strains containing the plasmids were plated In the light of the observations that host on selective L agar, single colonies suspended in nonserecombination systems are involved in the lective L broth (4) and immediately diluted in fresh L broth to lo4 cells/ml and incubated at 37°C. These cul- maintenance of ColEI (13) and CloDF 13 (14) tures, after appropriate dilution, were further subcultured plasmids, it was thought appropriate to study at 12-hintervals and dilutions were plated on nonselective the effectsof specific recombination mutations L agar plates for viable counting. After overnight incubation of the plates at 37”C, the plates with 100-500 on maintenance of pRS1708 and pRS2062. colonies were replica plated on suitable selective media The rates of loss of pACYCl84 from poputo determine the proportion of antibiotic resistant cells. lations of the recA-I3 strain AB2463 (1 Z), and Strains carrying pRS1708 (0) were resistant to chlor- the r-e&-21, recC-22 strain JC5519 (15), did amphenicol (25 pg ml-‘), strains carrying pRSl705 (m) not differ from that observedwith the isogenic and pRS2062 (V) were resistant to tetracycline (10 pg ml-‘), and those carrying pACYCl84 (0) were resistant ret+ strain (Fig. 2). On the other hand, to both antibiotics. Generation time was computed from pRS2062 was lost at a very rapid rate from the viable counts and 12-hcorrespondedto approximately cultures of each of the two recombination10 doublings for each of the strains used. Stability of the deficient strains (Figs. 2B and C). In recA muplasmids were determined in the ret+ strain ABl157 (A) tant cells, both the RecF and RecBC pathways and its isogenic derivatives, the recA-13 strain AB2463 (B), and the recE-21, recC-22 strain JC5519 (C). Each of recombination are inoperative, whereas in point denotesan averageof at least two independent mea- a recBC mutant, the RecBC pathway is surements. blocked but the RecF pathway remains intact
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(26). On this basis,our data may be interpreted to indicate that stabilization of the Par- pl5A replicon by the 56.4-64.7F segment of the leading region is mediated via the RecBC pathway of recombination which requires both the recA and recBC gene products. However, the pleiotropic nature of the RecA protein leaves open the possibility of other interpretations. Maintenance of pRS 1708, was not influenced by the recA mutation (Fig. 2B) and only a small, but reproducible, instability was apparent in the recBC mutant strain (Fig. 2C). These data, taken together with the results above, would suggestthat the presence of the 64.7-66.7F segmentoverrides the requirement of at least the RecA protein for stabilization of the pACYCl84 replicon by the 56.4-64.7F segment of the leading region. Alternatively, the absence of the 53.8-56.4F region from pRS1708 may bypass this requirement. Further evidence against the involvement of plasmid copy number in the stabilization of these recombinant plasmids is provided by our observation that the copy numbers of most of them did not differ significantly in the recA mutant strain from those in the ret+ strain. pACYC 184 was present at 22 copies per genome equivalent in each of the two recombination-deficient strains, while pRS 1708 had 7.8 and 3.1 copies in the recA and recBC strains, respectively, compared to 8.5 copies in the ret+ strain. pRS2062 had a copy number of 7.3 in the recA strain but 14 in the recBC strain. Thus, the twofold increase in the copy number of pRS2062 did not lead to a stable inheritance of the plasmid by the recBC mutant strain, nor was the instability of pRS2062 in the recA strain due to a decreased copy number. However, the approximately threefold reduction in the copy number of pRS 1708 in the recBC mutant could explain the reduced stability exhibited by pRS1708 in that strain (Fig. 2C). In order to examine the effect of the 64.766.7F segment on maintenance of the Parpl5A replicon in the absence of the 56.464.7F segment, the segregation kinetics of pRS1705 (64.7-73.0F EcoRI fragment f6 of F cloned into pACYC 184, Fig. 1) were measured in all three strains (Fig. 2). The results,
while clearly indicating that this segment of the leading region is not sufficient by itself to bring about the maintenance function, do not exclude the possibility that this region overrides the RecA dependenceof the f3-mediated Par function as suggestedabove. In this connection, it should be emphasized that the stability effect discussed in this communication is not related to the orientation-dependent instability of oriT+ clones in F+ cells asdescribed by Everett and Willetts (17). First, our experiments were performed in F cells and secondly, the difference in the stability of pRSl705 and pRSl708 could not be due to an orientation-dependent oriT effect; oriT is inserted in the same relative orientation with respectto the direction of replication from the p15A origin in both plasmids. The leading region segment between 56.4F and 66.7F codes for at least five polypeptides of molecular weights 33,400, 27,800, 23,100, 14,400, and 11,000 (4). A protein similar in size and map location to the 23,100-Da polypeptide has recently been shown (18) to be a single-stranded DNA (ssDNA) binding protein, the product of the ssfgene located at the 59.3F BumHI site on the leading region (Fig. 1). The 33,400-Da polypeptide, which is considered to be the 6d protein described earlier (19) and a 13,500-Da protein which was tentatively identified before (4), have now been mapped between 64.7F and 66.6F although their promoter is thought to be located on f3 (20). Functions of these proteins, and of the three remaining polypeptides, are not known. In support of the role of a f3-encoded protein in the stabilization of pACYC 184, preliminary experiments have shown that pACYC 184 was retained at nearly 100% level over 60 generations of nonselective growth by AB 1157 cells which additionally carried EcoRI f3 cloned in the compatible vector RSF2 124 (21) but not by ABI 157 which carried the vector RSF2124 alone. Such a result indicates that the D-mediated Par function acts in trans. The E. coli ssDNA binding protein (a product of the chromosomally encoded ssb gene), besides being essential for E. co/i DNA replication (22) is known to modulate the action of several enzymes such as the RecA protein
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(23) and the RecBC enzyme (exonuclease V) (24). Similarity in the nucleotide sequences of the s.sband the F-specified &genes has prompted the speculation that the properties of the proteins they encode may also be similar (25). On this basis, and in the light of our observation that f3-specified maintenance function is dependent on RecA and RecBC proteins in the absenceof the 64.7-66.6F segment, it should be interesting to investigate the role of the ssf and ssb genes in plasmid maintenance. It is, of course, potentially possible that any of the remaining three polypeptides of f3 are involved and we are also testing the possibility that a polypeptide coded by the 64.7-66.6F segment rescuesthe RecA dependence of the maintenance function. In this communication we have described the maintenance of the normally unstable pACYC184 replicon by the leading region of F but have not attempted to determine whether this activity plays a role in the stable maintenance of the F plasmid itself. While a par region involved in the partitioning and maintenance of F has been mapped (26,27) within the primary F replication-incompatibility region (Fig. l), the leading region may well provide a secondary F Par function. It may, on the other hand, be involved in posttransfer repliconation/maintenance of the F DNA in the recipient cell. ACKNOWLEDGMENTS We thank Louise O’Gorman and Robert Cross for technical assistance, David Cram for useful discussions, Barbara Bachmann (E. coli Genetic Stock Center, Yale University School of Medicine) for strains carrying ret mutations, and K. Brooks Low for communicating results prior to publication. This work was supported, in part, by a grant from the Australian Research Grants Scheme; A.R. was supported by a Monash Postgraduate Scholarship.
REFERENCES 1. WILLEITS, N., AND SKURRAY, R., Annu. Rev. Genet. 14,41-76 (1980). 2. KOMAI, N., NISHIZAWA, T., HAYAKAWA, Y., MuROT%, T., AND MAT~UBARA, K., Mol. Gen. Genet. 186, 193-203 (1982).
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