0021-9193/78/0133433$02.00/0
Vol. 133, No. 1 Printed in U.S.A.
JouRNAL OF BACTEOLOGY, Jan. 1978, p. 433-436 Copyright ) 1978 American Society for Microbiology
ColEl Plasmid Mutants Affecting Growth of an Escherichia coli recB recC sbcB Mutant JOSEPH INSELBURG Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755
Received for publication 15 August 1977 Three mutant derivatives of the plasmid ColEl were found to reduce the formation of plasmidless cells in a recB recC sbcB cell population. An active plasmid role in the plaid-host interaction is suggested.
Vapnek et al. (11) and Ream et al. (8; personal communication) have recently observed that the plasmid CoIEl is not stably maintained in a recB recC sbcB mutant of Escherichia coli that is deficient in exonuclease V (3, 9) and exonuclease I (7). An analysis of previously isolated ColEl mutants was undertaken to determine whether a plasmid function affected its maintenance in the recB recC sbcB host. Previous findings that ColEl plmmid-coded proteins are not essential for in vitro plsmid replication (10) and that as little as 16% of the CoIEl genome can be stably maintained in E. coli (6) suggested that the ColEl plasmid is passively maintained in the host. This study shows that the formation of plasmidless cells in the JC7623 recB recC sbcB cell population principally occurs during early cell growth in liquid culture and that their proportion is enhanced by both the death and an increased generation time of plasmid-carrying cells. Since three plasmid mutants are identified that suppress the plasmid-associated reduction in generation time of the host, JC7623, an active plasmid role in the plasmid-host interaction is suggested. E. coli JC7623, used by Ream et al. (8) to demonstrate CoE1 plmid instability in recB recC sbcB bacteria, was transformed with wildtype ColEl or a variety of CoIEl plasmid DNAs that contain the TnA transposon (5, 6; Table 1). The plmnid-carrying cells were selected as ampicillin-resistant (Apr) colonies or for ColEl as colicin-immune colonies. Whereas most Apr transformants produced microcolonies on Apcontaining agar, three produced "large" colonies simila in size to those of JC7623 grown on nutrient agar. Growth of single colonies of JC7623 Apr transformants in L-broth without Ap for 25 generations indicated that a strong correlation exists between the relative size of the transformant colony on Ap agar and the
appearance of plasmidless cells in culture (Table 1). No detectable plasmidless progeny were produced after 32 generations of growth in L-broth by P678-54 recB+ recC+ sbcB+ cells carrying any of the plasmids (data not shown). Cells carrying plasmid 6-30 (5), a deletion mutant derived from it, 6-30:d6-3 (6), and dO-11 (6) exhibited much lower levels of plasmidless progeny formation than wild-type ColEl or other ColEl mutants in the recB recC sbcB host (Table 1). The formation of plasmidless cells from single, transformed colonies was further studied by following the production of Ap8 or colicin-deficient cells during 30 h of logarithmic cell growth. Cells were serially diluted when they reached between 1 x 108 and 5 x 108 cells per ml. The growth curves of some transformants that either produced microcolonies or "large," normal-size colonies on nutrient-Ap agar are shown in Fig. 1. Those results (Fig. 1, Table 2) show that: (i) plasmidless cells were produced and survived in the environment of the colony grown on Ap agar; (ii) by 1 h of growth in L-broth, most cultures contained a high proportion of plasmidless cells even if the proportion in the original colony was low, suggesting that cells contain relatively few plasmid copies if plasmid segregation is random during cell division; (iii) during the first 3 to 6 h of growth of cells inoculated from nutrient agar to L-broth, there was appreciable cell death (recB recC celLs may show cell death [1, 12], but no simple explanation of this phenomenon exists); and (iv) except for celLs carrying one of the three mutant plasmids, 630, 6-30:d6-3, or dO-11 (Fig. 1, Table 2), the growth rate of cells containing the CoIEl plasmids is gificantly slower than the plasmidless JC7623 progeny derived from them. It is by this suppression of the plasmid's effect on the growth rate of the recB recC sbcB host that the mutations in plids 6-30, 6-30:d6-3, and dO-1l prevent an increase in the proportion of the plas-
433
434
J. BACTEFRIOL.
NOTES
TABLE3 1. Formation ofpkwsnidless cels from ColEl Apr and ColEl ransformants of JC7623a
PlasMidb Total
CoIEl TnA insertion: 6-30c 7-12c 2-14 0-1 3-12 3 lc 7-6 2-35c 5-42 6-12
Approx 25 generations
Time zero
1X
Col+
loll
1X lo05
Total
Ap'
3.5 X 106 1.3 X 106
3.8 x 106 1.2 X 10 _
_d _ 3.0 x 107 7.4 x 107 1.8 x 107
CoIEl deletion: Total 1.9 x l0o 6-30:d6-6c 5.7 x 106 6-30:d2-3 6-30.dl-4 8.0 x 103 6-30:d64 3.2 x 106 6-30:d6-1 6-30:d6-3c 6-30:d3-1 6-30:d2-7 d5-36 1.3 x 107 dO-llc
2.0 x 107
2.8 x 106 8.4 x 106
Ap' 2.1 x 10" 2.4 x 106 7.0 x 103 2.2 x 106 -
-
1.2 x 107
Total 1X109
Total 2.3 X 109 6.1 X 10"
ny size on Ap aar
Col+ 4 X 106
Apr 1.4 X 109
Vol. 133, No. 1 Printed in U.S.A.
JouRNAL OF BACTEOLOGY, Jan. 1978, p. 433-436 Copyright ) 1978 American Society for Microbiology
ColEl Plasmid Mutants Affecting Growth of an Escherichia coli recB recC sbcB Mutant JOSEPH INSELBURG Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755
Received for publication 15 August 1977 Three mutant derivatives of the plasmid ColEl were found to reduce the formation of plasmidless cells in a recB recC sbcB cell population. An active plasmid role in the plaid-host interaction is suggested.
Vapnek et al. (11) and Ream et al. (8; personal communication) have recently observed that the plasmid CoIEl is not stably maintained in a recB recC sbcB mutant of Escherichia coli that is deficient in exonuclease V (3, 9) and exonuclease I (7). An analysis of previously isolated ColEl mutants was undertaken to determine whether a plasmid function affected its maintenance in the recB recC sbcB host. Previous findings that ColEl plmmid-coded proteins are not essential for in vitro plsmid replication (10) and that as little as 16% of the CoIEl genome can be stably maintained in E. coli (6) suggested that the ColEl plasmid is passively maintained in the host. This study shows that the formation of plasmidless cells in the JC7623 recB recC sbcB cell population principally occurs during early cell growth in liquid culture and that their proportion is enhanced by both the death and an increased generation time of plasmid-carrying cells. Since three plasmid mutants are identified that suppress the plasmid-associated reduction in generation time of the host, JC7623, an active plasmid role in the plasmid-host interaction is suggested. E. coli JC7623, used by Ream et al. (8) to demonstrate CoE1 plmid instability in recB recC sbcB bacteria, was transformed with wildtype ColEl or a variety of CoIEl plasmid DNAs that contain the TnA transposon (5, 6; Table 1). The plmnid-carrying cells were selected as ampicillin-resistant (Apr) colonies or for ColEl as colicin-immune colonies. Whereas most Apr transformants produced microcolonies on Apcontaining agar, three produced "large" colonies simila in size to those of JC7623 grown on nutrient agar. Growth of single colonies of JC7623 Apr transformants in L-broth without Ap for 25 generations indicated that a strong correlation exists between the relative size of the transformant colony on Ap agar and the
appearance of plasmidless cells in culture (Table 1). No detectable plasmidless progeny were produced after 32 generations of growth in L-broth by P678-54 recB+ recC+ sbcB+ cells carrying any of the plasmids (data not shown). Cells carrying plasmid 6-30 (5), a deletion mutant derived from it, 6-30:d6-3 (6), and dO-11 (6) exhibited much lower levels of plasmidless progeny formation than wild-type ColEl or other ColEl mutants in the recB recC sbcB host (Table 1). The formation of plasmidless cells from single, transformed colonies was further studied by following the production of Ap8 or colicin-deficient cells during 30 h of logarithmic cell growth. Cells were serially diluted when they reached between 1 x 108 and 5 x 108 cells per ml. The growth curves of some transformants that either produced microcolonies or "large," normal-size colonies on nutrient-Ap agar are shown in Fig. 1. Those results (Fig. 1, Table 2) show that: (i) plasmidless cells were produced and survived in the environment of the colony grown on Ap agar; (ii) by 1 h of growth in L-broth, most cultures contained a high proportion of plasmidless cells even if the proportion in the original colony was low, suggesting that cells contain relatively few plasmid copies if plasmid segregation is random during cell division; (iii) during the first 3 to 6 h of growth of cells inoculated from nutrient agar to L-broth, there was appreciable cell death (recB recC celLs may show cell death [1, 12], but no simple explanation of this phenomenon exists); and (iv) except for celLs carrying one of the three mutant plasmids, 630, 6-30:d6-3, or dO-11 (Fig. 1, Table 2), the growth rate of cells containing the CoIEl plasmids is gificantly slower than the plasmidless JC7623 progeny derived from them. It is by this suppression of the plasmid's effect on the growth rate of the recB recC sbcB host that the mutations in plids 6-30, 6-30:d6-3, and dO-1l prevent an increase in the proportion of the plas-
433
434
J. BACTEFRIOL.
NOTES
TABLE3 1. Formation ofpkwsnidless cels from ColEl Apr and ColEl ransformants of JC7623a
PlasMidb Total
CoIEl TnA insertion: 6-30c 7-12c 2-14 0-1 3-12 3 lc 7-6 2-35c 5-42 6-12
Approx 25 generations
Time zero
1X
Col+
loll
1X lo05
Total
Ap'
3.5 X 106 1.3 X 106
3.8 x 106 1.2 X 10 _
_d _ 3.0 x 107 7.4 x 107 1.8 x 107
CoIEl deletion: Total 1.9 x l0o 6-30:d6-6c 5.7 x 106 6-30:d2-3 6-30.dl-4 8.0 x 103 6-30:d64 3.2 x 106 6-30:d6-1 6-30:d6-3c 6-30:d3-1 6-30:d2-7 d5-36 1.3 x 107 dO-llc
2.0 x 107
2.8 x 106 8.4 x 106
Ap' 2.1 x 10" 2.4 x 106 7.0 x 103 2.2 x 106 -
-
1.2 x 107
Total 1X109
Total 2.3 X 109 6.1 X 10"
ny size on Ap aar
Col+ 4 X 106
Apr 1.4 X 109
