- Email: [email protected]
Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli
J. nlol.
BiOl.
(1974) 89, 647-650
Electron Microscope Heteroduplex Studies of Sequence Relations Among Plasmids of Esckerichia coli IX.1 Note on the Deletion Mutant of F, FA(33-43) WILLIAM &I. AXTHONY, RICH&D
C. DEONIER$, HUNG-JUNG
EIICHI OHTSUEO$, NORMAX
LEE, SYLVIA Hu,
DAVIDSON
Department of Chemistry Institute of Technology Pasadena, Calif. 91109, U.S.A.
California
AND
PAUL BRODA Medical
Research Council Molecular Genetics Unit Department of Molecular Biology University of Edinburgh, King’s Buildings MayJield Road, Edinburgh EH9 3JR, Xcotland (Received 1 April
197’4)
Heteroduplex analysis shows that the plasmid extracte’d from cells of the F’ strain, W1655, is a deletion mutant of F, and lacks segments with F co-ordinates 32.6 to 42.9 kilobases. The genes on F for resistance to the female-specific phages T3, 411, and 7 probably lie within this region. Consideration of the sequences deleted in several F-primes shows that the sequences from 0 to 42.9 kilobases on F are not necessary for autonomous replication nor for fertility.
1. Introduction We wish to report that the F plasmid present in cells of an F + strain, W1655, used in some previous studies (Broda, 1967) is a deletion mutant of F. It is missing the sequences of F from 32.6 to 42.9 kb // in the co-ordinate system that we have introduced for the F factor (Sharp et al., 1972), and is therefore designated FA(33-43). It is useful as a reference molecule in heteroduplex studies of other F-primes.
2. Materials and Methods Strain W1655, which is m&B-h-X, was derived from .EEschwichia coli K12 in a series of steps, some of which were mutagenic (see chart 5 of Bachmann, 1972). It carries a transmissible F plasmicl that is mutant, in that it does not confer upon its host resistance to t This is one of a series of accompanying papers. Paper VIII is Ohtsubo et al. (19746). $ Present address: Department of Chemistry, University of Southern California, University Park, Los Angeles, Calif. 90007, U.S.A. 5 Present address: Department of Microbiology, University of New York at Stony Brook, Stony Brook, N.Y. 11790, U.S.A. jl Abbreviation used: kb, kilobases, t-housands of bases or base-pairs for single strand and duplex DNA, respectively. 647
648
W.
ET
M. ANTHONY
AL.
phage T3, which plates with low efficiency on most strains carrying F (Schell et al., 1963)t phage 411 and phage 7 (see Results). JE3100, bearing the plasmid F8-2, has been described elsewhere. JE2571 is the isogenic F- progenitor of JE3100 (Ohtsubo et al., 1970; Sharp et al., 1972; Ohtsubo & Hsu, 1974). JE102 F- is a lysogen of phage 7 (Hakura et al., 1964). The methods of plasmid extraction and electron microscopy that we used have been described by Sharp et al. (1972) and Ohtsubo et al. (1974). Sensitivity to phage 7 was tested as follows. Several &ml samples of a T-broth culture of JE102 at a cell density of 1.5 x lo7 cells/ml were induced by exposure to U.V. light and incubated at 37°C until the onset of lysis. The remaining bacteria were lysed by adding chloroform. Dilutions (1: 10, 1:50, and 1: 100) of the lysates were streaked on T-broth agar plates. The bacteria to be tested were then cross-streaked on the plates.
3. Results In ant’icipation of the results reported below, we designate the plasmid extracted from W16.55 as F~l(33-43). Its length, as measured by spreading the open circular DNA by the aqueous technique, was observed to be shorter than that of our standard F from W1485 by lOf2%. A careful, direct comparison with F gives a value of OSS7-&0*016 for the length ratio, suggesting that the molecular weight of FA(33-43) is 83Skl.6 kb. (F is taken as 94.5 kb (Sharp et al., 1972).)
32 Sk,‘42 (ai
9F
-.-
FA(33-431 F 5CF
jj ’ “,5OF
I 94 55’Ci 8 YF- I6 3F
9’ “f ps.5i,‘or
32’61
42 !rF
32 (SF,‘42
91.
Cl (bl
FA133-431 FE-33
d , 6 iidk-22Ckb-d L
50F
I \\
-4 ‘\
,’
,“.
.:OF
‘Y
429F .f’;%F,
326F 94 Sl-,‘dF
,,Y 8F
\
32 6F/42
OF ic
F
[cl
FA133-43; FIZZ-i
L--p50F
i
L----_, 2 VP/-(
L ’ .X’ 61
‘\yF <:,j ‘2,
FIG. 1. Heteroduplexes used for the structure determination of FA(33-43) as discussed in the text. Note that circular molecules are represented in a linear form by cutting the duplex at the point SOF, which lacks interesting features.
The structure of the plasmid was deduced from a study of its heteroduplexes with F and with the reference F-primes, FS-33 and F152-1. The structures of these latter two reference F-primes are described elsewhere (Ohtsubo & Hsu, personal communication; Sharp et al., 1972). The observed structures of the heteroduplexes of Fd (33-43) are depicted in Figure 1. Electron micrographs of heteroduplexes with F and with F152-1 are shown in Plate I. The structure observed for the F/FA(33-43) heteroduplex shows that the latter molecule is a simple deletion mutant of F, but it does not map the position of the t Strain W1655 was referred to mistakenly by &hell el al. (1963: Table 1, cross number 10) as strain W1486. The F factor from the true strain W1486 is not deleted in the 33-43 kb region (Sharp et aZ., 1972; and Results). Strain WI655 from the laboratory of Broda is As. The original Wl656 from the Lederberg laboratory is As (B. Bachmann, personal communication).
hATE
duplex $X174 dards.
I (a) Fd(33-43)/F heteroduplex as depicted in Fig. l(a). (b) FA(33-43)/F152-1 heteroits depicted in Fig. l(c). Various features of the molecules are marked on the micrographs. single-stranded and double-stranded molecules are present and were used as length stanTheir length is approximately 5.1 lib. [facing
1). Cd3
DELETION
MUTANT
OF F
649
deletion. Direct measurements of the single-strand loop indicate that the deletion has a length of 10&l kb. In the F8-33/FA(33-43) heteroduplex, the 33-43 deletion loop is mapped at approximately 17 kb to the right of the point 16*3F, which marks the clockwise terminus of the F&33/F non-homology substitution loop. The clockwise direction is demonstrated by the position of a little loop, c, at 91*OF kb. Thus the deletion has co-ordinates of approximately 33 to 43F kb. The structure is confirmed by the FL%l/FA(33-43) heteroduplex. In this case the 33-43 deletion loop is observed to be 29.7 kb displaced from one terminus of the F152-l/F substitution loop. From this structure alone, one cannot tell whether the deletion is clockwise or counterclockwise relative to the Fl52-l/F substitution feature, but this ambiguity is resolved by the FS-33/FA(33-43) heteroduplex described above. The co-ordinates of the deleted region can be calculated from measurements of the duplex lengths between the 33-43 deletion loop and the several non-homology features of the reference F-primes. The total length of the deleted region can be obtained by measuring the single-strand length of the deletion loop and, independently, as a difference between the duplex lengths of F and FA(33-43). We also take the length of standard F as 94.5 kb. An analysis of all the data on FA(33-43) heteroduplexes gives co-ordinates of 32.642.9F for the deleted region. Therefore, the calculated value for the length of FA(33-43) is 84.3fl.O kb. This is consistent with the molecular weight (836 f 1.6 kb) described above. We observe that E. coli W1655 carrying FA(33-43) is sensitive to phage 7 by the tests described in Materials and Methods. The control tests with the typical male strain, JE3100 carrying an F gal, F8-2, and the isogenic F- strain, JE2571, gave the expected results that the former was not sensitive to phage 7, whereas the latter was.
4. Discussion FA(33-43) is an additional useful reference F for heteroduplex mapping of other F-primes. Its application for these purposes is illustrated in our previous papers (Ohtsubo et al., 1974a). The F-prime factors derived from FlOO and F152 are deleted in the F sequences 0 to 28F (Ohtsubo & Hsu, 1974). The F8 episomes are deleted in the F sequences 8.5 to 16.3 kb. There is a deletion mutant of F, FA(O-15.0) that is deleted in the sequences indicated (previously called FA(O-14.5)) (Sharp et aE., 1972). The F-prime F210, is deleted in the sequences 8.5 to 115F. The plasmid F13-4 is deleted in the F intervals 16.3 to 37.2 kb (S. Hu & E. Ohtsubo, personal communication). The F’ilv F16 is deleted in the sequences 8.5 to 358F (Lee et al., 1974). All of these F and Fprimes are fertile (tra+) and capable of autonomous replication, as is FA(33-43). Thus, the sequences of F from 0 to 42.9 are not essential for these functions. As discussed previously, the region from 0 to 16*3F seems to be involved in the recombination of F with the E. coli chromosome to form Hfr’s and in subsequent excisions to give F-primes (Sharp et al., 1972; Ohtsubo & Hsu, personal communication). Strain W1655 carrying FA(33-43) is sensitive to phages T3 (Schell et al., 1963), 411 (I?. Broda, personal communication) and 7 as reported here. Many strains carrying F or F-prime factors are resistant to these female-specific phages. Strain 58-161F+ is resistant to T3 (Schell et aZ., 1963). These observations are consistent with the hypothesis that the gene(s) conferring resistance to female-specific phages lie in the 33-43 region that is deleted in FA(33-43).
650
W.
M. ANTHONY
El’
AL.
This research has been supported by the United States Public Health Service by a research grant (no. GM10991) to one of us (N. D.), by support as trainees under GM01262 fellowship to another to other authors (W. M .A.) and (H. J. L.) and by a postdoctoral author (R. C. D.). This is contribution no. 4831 from the Department of Chemistry, California Institute of Technology, Calif., U.S.A. REFERENCES Bachmann, B. J. (1972). Bacterial. Rev. 36, 525-557. Broda, P. (1967). Genet. Res. Camb. 9, 35-47. Hakura, A., Otsuji, N. & Hirota, Y. (1964). J. Gem. Miwobiol. 35, 69-73. Lee, H. J., Ohtsubo, E., Deonier, R. C. & Davidson, N. (1973). J. MoZ. Biol. 89, 585-597. Ohtsubo, E., Nishimura, Y. & Hirota, Y. (1970). Genetics, 64, 173-188. Ohtsubo, E., Deonier, R. C., Lee, H. J. & Davidson, N. (1974a). J. Mol. BioZ. 89, 565-584. Ohtsubo, E., Soll, L., Deonier, R. C., Lee, H. J. & Davidson, N. (19748). J. Mol. BioZ. 89, 631-646. Schell, J., Glover, S. W., Stacey, K. A., Broda, P. M. A. & Symonds, N. (1963). Genet. Res. Camb. 5,483-484. Sharp, P. A., Hsu, M. T., Ohtsubo, E. & Davidson, N. (1972). J. Mol. BioZ. 71,471-497.
BiOl.
(1974) 89, 647-650
Electron Microscope Heteroduplex Studies of Sequence Relations Among Plasmids of Esckerichia coli IX.1 Note on the Deletion Mutant of F, FA(33-43) WILLIAM &I. AXTHONY, RICH&D
C. DEONIER$, HUNG-JUNG
EIICHI OHTSUEO$, NORMAX
LEE, SYLVIA Hu,
DAVIDSON
Department of Chemistry Institute of Technology Pasadena, Calif. 91109, U.S.A.
California
AND
PAUL BRODA Medical
Research Council Molecular Genetics Unit Department of Molecular Biology University of Edinburgh, King’s Buildings MayJield Road, Edinburgh EH9 3JR, Xcotland (Received 1 April
197’4)
Heteroduplex analysis shows that the plasmid extracte’d from cells of the F’ strain, W1655, is a deletion mutant of F, and lacks segments with F co-ordinates 32.6 to 42.9 kilobases. The genes on F for resistance to the female-specific phages T3, 411, and 7 probably lie within this region. Consideration of the sequences deleted in several F-primes shows that the sequences from 0 to 42.9 kilobases on F are not necessary for autonomous replication nor for fertility.
1. Introduction We wish to report that the F plasmid present in cells of an F + strain, W1655, used in some previous studies (Broda, 1967) is a deletion mutant of F. It is missing the sequences of F from 32.6 to 42.9 kb // in the co-ordinate system that we have introduced for the F factor (Sharp et al., 1972), and is therefore designated FA(33-43). It is useful as a reference molecule in heteroduplex studies of other F-primes.
2. Materials and Methods Strain W1655, which is m&B-h-X, was derived from .EEschwichia coli K12 in a series of steps, some of which were mutagenic (see chart 5 of Bachmann, 1972). It carries a transmissible F plasmicl that is mutant, in that it does not confer upon its host resistance to t This is one of a series of accompanying papers. Paper VIII is Ohtsubo et al. (19746). $ Present address: Department of Chemistry, University of Southern California, University Park, Los Angeles, Calif. 90007, U.S.A. 5 Present address: Department of Microbiology, University of New York at Stony Brook, Stony Brook, N.Y. 11790, U.S.A. jl Abbreviation used: kb, kilobases, t-housands of bases or base-pairs for single strand and duplex DNA, respectively. 647
648
W.
ET
M. ANTHONY
AL.
phage T3, which plates with low efficiency on most strains carrying F (Schell et al., 1963)t phage 411 and phage 7 (see Results). JE3100, bearing the plasmid F8-2, has been described elsewhere. JE2571 is the isogenic F- progenitor of JE3100 (Ohtsubo et al., 1970; Sharp et al., 1972; Ohtsubo & Hsu, 1974). JE102 F- is a lysogen of phage 7 (Hakura et al., 1964). The methods of plasmid extraction and electron microscopy that we used have been described by Sharp et al. (1972) and Ohtsubo et al. (1974). Sensitivity to phage 7 was tested as follows. Several &ml samples of a T-broth culture of JE102 at a cell density of 1.5 x lo7 cells/ml were induced by exposure to U.V. light and incubated at 37°C until the onset of lysis. The remaining bacteria were lysed by adding chloroform. Dilutions (1: 10, 1:50, and 1: 100) of the lysates were streaked on T-broth agar plates. The bacteria to be tested were then cross-streaked on the plates.
3. Results In ant’icipation of the results reported below, we designate the plasmid extracted from W16.55 as F~l(33-43). Its length, as measured by spreading the open circular DNA by the aqueous technique, was observed to be shorter than that of our standard F from W1485 by lOf2%. A careful, direct comparison with F gives a value of OSS7-&0*016 for the length ratio, suggesting that the molecular weight of FA(33-43) is 83Skl.6 kb. (F is taken as 94.5 kb (Sharp et al., 1972).)
32 Sk,‘42 (ai
9F
-.-
FA(33-431 F 5CF
jj ’ “,5OF
I 94 55’Ci 8 YF- I6 3F
9’ “f ps.5i,‘or
32’61
42 !rF
32 (SF,‘42
91.
Cl (bl
FA133-431 FE-33
d , 6 iidk-22Ckb-d L
50F
I \\
-4 ‘\
,’
,“.
.:OF
‘Y
429F .f’;%F,
326F 94 Sl-,‘dF
,,Y 8F
\
32 6F/42
OF ic
F
[cl
FA133-43; FIZZ-i
L--p50F
i
L----_, 2 VP/-(
L ’ .X’ 61
‘\yF <:,j ‘2,
FIG. 1. Heteroduplexes used for the structure determination of FA(33-43) as discussed in the text. Note that circular molecules are represented in a linear form by cutting the duplex at the point SOF, which lacks interesting features.
The structure of the plasmid was deduced from a study of its heteroduplexes with F and with the reference F-primes, FS-33 and F152-1. The structures of these latter two reference F-primes are described elsewhere (Ohtsubo & Hsu, personal communication; Sharp et al., 1972). The observed structures of the heteroduplexes of Fd (33-43) are depicted in Figure 1. Electron micrographs of heteroduplexes with F and with F152-1 are shown in Plate I. The structure observed for the F/FA(33-43) heteroduplex shows that the latter molecule is a simple deletion mutant of F, but it does not map the position of the t Strain W1655 was referred to mistakenly by &hell el al. (1963: Table 1, cross number 10) as strain W1486. The F factor from the true strain W1486 is not deleted in the 33-43 kb region (Sharp et aZ., 1972; and Results). Strain WI655 from the laboratory of Broda is As. The original Wl656 from the Lederberg laboratory is As (B. Bachmann, personal communication).
hATE
duplex $X174 dards.
I (a) Fd(33-43)/F heteroduplex as depicted in Fig. l(a). (b) FA(33-43)/F152-1 heteroits depicted in Fig. l(c). Various features of the molecules are marked on the micrographs. single-stranded and double-stranded molecules are present and were used as length stanTheir length is approximately 5.1 lib. [facing
1). Cd3
DELETION
MUTANT
OF F
649
deletion. Direct measurements of the single-strand loop indicate that the deletion has a length of 10&l kb. In the F8-33/FA(33-43) heteroduplex, the 33-43 deletion loop is mapped at approximately 17 kb to the right of the point 16*3F, which marks the clockwise terminus of the F&33/F non-homology substitution loop. The clockwise direction is demonstrated by the position of a little loop, c, at 91*OF kb. Thus the deletion has co-ordinates of approximately 33 to 43F kb. The structure is confirmed by the FL%l/FA(33-43) heteroduplex. In this case the 33-43 deletion loop is observed to be 29.7 kb displaced from one terminus of the F152-l/F substitution loop. From this structure alone, one cannot tell whether the deletion is clockwise or counterclockwise relative to the Fl52-l/F substitution feature, but this ambiguity is resolved by the FS-33/FA(33-43) heteroduplex described above. The co-ordinates of the deleted region can be calculated from measurements of the duplex lengths between the 33-43 deletion loop and the several non-homology features of the reference F-primes. The total length of the deleted region can be obtained by measuring the single-strand length of the deletion loop and, independently, as a difference between the duplex lengths of F and FA(33-43). We also take the length of standard F as 94.5 kb. An analysis of all the data on FA(33-43) heteroduplexes gives co-ordinates of 32.642.9F for the deleted region. Therefore, the calculated value for the length of FA(33-43) is 84.3fl.O kb. This is consistent with the molecular weight (836 f 1.6 kb) described above. We observe that E. coli W1655 carrying FA(33-43) is sensitive to phage 7 by the tests described in Materials and Methods. The control tests with the typical male strain, JE3100 carrying an F gal, F8-2, and the isogenic F- strain, JE2571, gave the expected results that the former was not sensitive to phage 7, whereas the latter was.
4. Discussion FA(33-43) is an additional useful reference F for heteroduplex mapping of other F-primes. Its application for these purposes is illustrated in our previous papers (Ohtsubo et al., 1974a). The F-prime factors derived from FlOO and F152 are deleted in the F sequences 0 to 28F (Ohtsubo & Hsu, 1974). The F8 episomes are deleted in the F sequences 8.5 to 16.3 kb. There is a deletion mutant of F, FA(O-15.0) that is deleted in the sequences indicated (previously called FA(O-14.5)) (Sharp et aE., 1972). The F-prime F210, is deleted in the sequences 8.5 to 115F. The plasmid F13-4 is deleted in the F intervals 16.3 to 37.2 kb (S. Hu & E. Ohtsubo, personal communication). The F’ilv F16 is deleted in the sequences 8.5 to 358F (Lee et al., 1974). All of these F and Fprimes are fertile (tra+) and capable of autonomous replication, as is FA(33-43). Thus, the sequences of F from 0 to 42.9 are not essential for these functions. As discussed previously, the region from 0 to 16*3F seems to be involved in the recombination of F with the E. coli chromosome to form Hfr’s and in subsequent excisions to give F-primes (Sharp et al., 1972; Ohtsubo & Hsu, personal communication). Strain W1655 carrying FA(33-43) is sensitive to phages T3 (Schell et al., 1963), 411 (I?. Broda, personal communication) and 7 as reported here. Many strains carrying F or F-prime factors are resistant to these female-specific phages. Strain 58-161F+ is resistant to T3 (Schell et aZ., 1963). These observations are consistent with the hypothesis that the gene(s) conferring resistance to female-specific phages lie in the 33-43 region that is deleted in FA(33-43).
650
W.
M. ANTHONY
El’
AL.
This research has been supported by the United States Public Health Service by a research grant (no. GM10991) to one of us (N. D.), by support as trainees under GM01262 fellowship to another to other authors (W. M .A.) and (H. J. L.) and by a postdoctoral author (R. C. D.). This is contribution no. 4831 from the Department of Chemistry, California Institute of Technology, Calif., U.S.A. REFERENCES Bachmann, B. J. (1972). Bacterial. Rev. 36, 525-557. Broda, P. (1967). Genet. Res. Camb. 9, 35-47. Hakura, A., Otsuji, N. & Hirota, Y. (1964). J. Gem. Miwobiol. 35, 69-73. Lee, H. J., Ohtsubo, E., Deonier, R. C. & Davidson, N. (1973). J. MoZ. Biol. 89, 585-597. Ohtsubo, E., Nishimura, Y. & Hirota, Y. (1970). Genetics, 64, 173-188. Ohtsubo, E., Deonier, R. C., Lee, H. J. & Davidson, N. (1974a). J. Mol. BioZ. 89, 565-584. Ohtsubo, E., Soll, L., Deonier, R. C., Lee, H. J. & Davidson, N. (19748). J. Mol. BioZ. 89, 631-646. Schell, J., Glover, S. W., Stacey, K. A., Broda, P. M. A. & Symonds, N. (1963). Genet. Res. Camb. 5,483-484. Sharp, P. A., Hsu, M. T., Ohtsubo, E. & Davidson, N. (1972). J. Mol. BioZ. 71,471-497.