JOLURNAL OF VIROLOCY, Feb. 1979, p. 808-81()
Vol. 29, No. 2
()()')'0)-5:38X/ 79/)/02-08()8/();3.$(',02.0()/()
Chromosomal Location of the Attachment Site for the PA-2 Prophage in Escherichia coli K-12 ANTHONY P. PUGSLEY,* DORIS LITTMANN-LOUTH, AND CARL A. SCHNAITMAN Department of Microbiology, University of Virginia Medical School, Charlottesville, Virginia 22908
Received for publication 9 August 1978
The chromosomal attachment site for the PA-2 prophage is located between dsd and aroC at approximately 50 min on the Escherichia coli K-12 genetic map. The attachment site is designated attPA-2.
Bacteriophage PA-2 lysogens of Escherichia coli K-12 produce substantial amounts of a new outer membrane protein (protein 2) which largely replaces two other major outer membrane proteins (la and lb [2, 5]). Proteins la, lb, and 2 all function as transmembrane pores or "porins" (5), and protein lb is also the receptor for bacteriphage PA-2 (2). PA-2 appears to be closely related to bacteriophage X, which it resembles morphologically. DNA from PA-2 and X can recombine, and a range of PA-2-X hybrids has been described (2). The availability of bacteriophage PA-2 hx (hy7 [2]), a derivative of hy7 carrying a mutation in a gene (Ic) controlling production of protein 2 (5), and a temperature-sensitive clear-plaque mutant of PA-2 that forms stable lysogens only at 30°C or lower (PA-2 ts9) has enabled us to locate the PA-2 attachment site (attPA-2) on the E. coli K-12 chromosome. Preliminary conjugation experiments with hy7 lysogens of a number of different Hfr strains and PA-2 ts9 lysogens of a number of F- strains indicated that attPA-2 was located between 48 and 53 min on the E. coli K-12 chromosome. This was confirmed by P1 transduction experiments (4) in which strain CS396 was used as the donor and strain CS419 as the recipient (see Table 1) with selection for ara + or pur+ recombinants at 30°C. Transductants were scored for ability to grow at 42°C, indicating transfer of the ts9+ marker on the prophage of the donor chromosome. Analysis of cotransduction frequencies and the different classes of recombinants (Table 2) indicated that the gene order is dsd-attPA2-aroC-purF. A comparison of the cotransduction frequencies between dsd and aroC in lysogenized and nonlysogenized strains (Table 2) indicated that the increase in the distance between the two markers in the lysogenized cells corresponded to an equivalent distance of 1.1 min of the E. coli K-12 chromosome or 44 kilobases (1), which is in close agreement with the known size of the A prophage (3).
To confirm that the identified attPA-2 locus is the preferred attachment site for the PA-2 prophage, we selected 20 independent PA-2 ts9 lysogens of strain EM3003 and used these as recipients in conjugation experiments. Strain CS411 was used as the donor with selection for aro+ recombinants in the presence of streptomycin (100 jig/ml) at 30°C as described by Miller (4). In each case, the majority of the recombinants were able to grow at 42°C indicating a high frequency of transfer of the ts9+ marker and close linkage between attPA-2 and aroC. In a further set of transduction experiments between CS396 and CS419, we also scored for transfer of the two other prophage markers, h (host range) and lc. The host range (h IPA-2 or h") of the progeny phage from the selected aroC+ recombinants was determined by assaying their ability to form plaques on strains CS208 (a Par mutant lacking the PA-2 receptor [2, 5]) and poplO75 (a LamB mutant lacking the A receptor). Production of protein 2 was determined by polyacrylamide gel electrophoresis. For this purpose we employed a novel membrane fractionation procedure in which very small volumes of cells are used. Cells growing rapidly in 6 ml of TY medium (5) containing 500 liM MgCl2 at 30°C (5 x 10' cells per ml) were treated with 5 ig of polymyxin B per ml for 10 min followed by 0.5% sodium dodecyl sulfate until lysis was complete (15 to 30 min). The cell debris (membrane fraction) was then pelleted by centrifugation at 46,000 x g for 10 min at 10°C and suspended in sample buffer for electrophoresis using the Trisglycine-hydrochloride buffer system described previously (2). This procedure produced a membrane fraction which contained some proteins from both the inner and outer membranes. The major outer membrane proteins 3a and 3b were not present, and proteins la, lb, and 2 were clearly resolved. The results of the transduction analysis (Table 2) indicate that the prophage markers ts9, lc, and h are all closely linked and that the lc 808
NOTES
VOL. 29, 1979
protein 2 is unknown at the present time. A chromosomal gene linked to par also appears to affect production of protein 2 (2). However, there does not appear to be a separate PA-2 gene controlling the levels of proteins la and lb in PA-2 lysogens. We have examined wild-type cells lysogenized with several independently isolated hy7lc phage and find that these strains produce normal (wild-type) levels of proteins la and lb. This presumably indicates that it is competition for a limited number of common potential insertion sites that regulates the number of copies of each of these proteins that are inserted into the membrane, and that synthesis of all three proteins is regulated by a feedback mechanism. Although PA-2 and A appear to be closely related, we have thus far been unable to demonstrate PA-2-mediated specialized transduc-
may be closer to dsd than the other two markers. We are thus able to define a region between dsd and aroC within which attPA-2 is situated (Fig. 1). The role of the Ic gene in the production of TABLE 1. Strains of E. coli K-12 Strain no. W 1485F CS208
pI)op105
CS:396
EM:3003 CS419 CS411
Relevant characteristics
thi pao- 10 thi HfrG6 lamnB204 h7l17c lsogen of W1485F
aog amoC porF thi dsdc lay iPsL
809
Source and reference C.G.S.C'. (3) M. Hofnung T'his paper (5) E. McFall This paper This paper
PA-2 ts9 lxsogen of EM,3003 HfrKI,16 thi-I -el- I thyA2'4 no/A 13 di-zz I'A-2 " Coli (;enetic Stock Center, New Haven. Cotnn.
TABLE 2. Transduction data
CS;39i (hv7c35)
Donor markers Selectedrnsdce transduced
Selected marker (colonies tested)
Recipient
P1 grown on
purF (272)
CS419 (put-F asoC dsd
CS396
Marker
No. )
PA-2 ts9'
8 (3) 116 (43)
aroC'
PA-2Ws9)
dsd' PA-2 t.(s9'
aro(C (27)))
CS419
co-
Frequency of recombinant classes purF
PA-2 (s9
dsd
+
+ +
+ +
+
-
No.
O
49 (18) 95 (35) 7 (3)
purE'
dsd5('
+
I 6
2)
+
W1485F
CS396
EM3(X)3 (purF aroC d/sd)
piiF'
ato' (1(X))
CS419
L-
L dsd
a)o()' (30)1)
C
1
1
1 17
purFE (Isd'
36
T'A-2 ts9' T'A-2 hA PA-2 1e
18
36 1 1
aroC
tS9
-
_
-
-
95 119
1II) (3)
t.sd'
1
0.87 1.41
+
(56)3
>
20
12
I 0.60 ] 0.50 J
purF
Lysogen
. a *~~~~~
%lb
I% %%
I
%;4~.-I1I %46
Non- lysogen
did
aioC
,0.34 L
puirF 0.58 X
FIG. 1. Detail of the linkage map of Escherichia coli K-12 in the region purF-dsd showing the region occupied by the PA-2 prophage. The shaded area represents the E. coli chromosome, and the hatched area represents a region of either E. coli or PA-2 DNA. The clear area represents the region that must be occupied by PA-2 DNA. Figures are map distances (in minutes) of the E. coli K-12 chromosome (1).
810
NOTES
tion of the aroC gene under conditions that work well for A-mediated transduction of gal (4). This work was supported by Public Health Service grant GM-18006 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Bachmann, B. J., K. B. Low, and A. L. Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 2. Bassford, P. J., D. L. Diedrich, C. A. Schnaitman, and P. Reeves. 1977. Outer membrane proteins of Escherichia coli. VI. Protein alteration in bacteriophage-resistant mutants. J. Bacteriol. 131:608-622.
J. VIROL. 3. Davidson, N., and W. Szybalski. 1971. Physical and chemical characteristics of lambda DNA, p. 45-82. In A. D. Hershev (ed.). The bacteriophage lambda. Cold Spring lHarborI Laboratory, Cold Spring Harbor. N.Y. 4. Miller, J. H. 1972. Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 5. Pugsley, A. P., and C. A. Schnaitman. 1978. Outer membrane proteins of Escherichia coli. VII. Evidence that bacteriophage-directed protein 2 functions as a pore. J. Bacteriol. 133:1181-1189. 6. Schnaitman, C. A., D. Smith, and M. F. deSalas. 1975. Temperate bacteriophage that causes production of a new major outer membrane protein in Escherichia coli. J. Virol. 15:1121-1130.
Vol. 29, No. 2
()()')'0)-5:38X/ 79/)/02-08()8/();3.$(',02.0()/()
Chromosomal Location of the Attachment Site for the PA-2 Prophage in Escherichia coli K-12 ANTHONY P. PUGSLEY,* DORIS LITTMANN-LOUTH, AND CARL A. SCHNAITMAN Department of Microbiology, University of Virginia Medical School, Charlottesville, Virginia 22908
Received for publication 9 August 1978
The chromosomal attachment site for the PA-2 prophage is located between dsd and aroC at approximately 50 min on the Escherichia coli K-12 genetic map. The attachment site is designated attPA-2.
Bacteriophage PA-2 lysogens of Escherichia coli K-12 produce substantial amounts of a new outer membrane protein (protein 2) which largely replaces two other major outer membrane proteins (la and lb [2, 5]). Proteins la, lb, and 2 all function as transmembrane pores or "porins" (5), and protein lb is also the receptor for bacteriphage PA-2 (2). PA-2 appears to be closely related to bacteriophage X, which it resembles morphologically. DNA from PA-2 and X can recombine, and a range of PA-2-X hybrids has been described (2). The availability of bacteriophage PA-2 hx (hy7 [2]), a derivative of hy7 carrying a mutation in a gene (Ic) controlling production of protein 2 (5), and a temperature-sensitive clear-plaque mutant of PA-2 that forms stable lysogens only at 30°C or lower (PA-2 ts9) has enabled us to locate the PA-2 attachment site (attPA-2) on the E. coli K-12 chromosome. Preliminary conjugation experiments with hy7 lysogens of a number of different Hfr strains and PA-2 ts9 lysogens of a number of F- strains indicated that attPA-2 was located between 48 and 53 min on the E. coli K-12 chromosome. This was confirmed by P1 transduction experiments (4) in which strain CS396 was used as the donor and strain CS419 as the recipient (see Table 1) with selection for ara + or pur+ recombinants at 30°C. Transductants were scored for ability to grow at 42°C, indicating transfer of the ts9+ marker on the prophage of the donor chromosome. Analysis of cotransduction frequencies and the different classes of recombinants (Table 2) indicated that the gene order is dsd-attPA2-aroC-purF. A comparison of the cotransduction frequencies between dsd and aroC in lysogenized and nonlysogenized strains (Table 2) indicated that the increase in the distance between the two markers in the lysogenized cells corresponded to an equivalent distance of 1.1 min of the E. coli K-12 chromosome or 44 kilobases (1), which is in close agreement with the known size of the A prophage (3).
To confirm that the identified attPA-2 locus is the preferred attachment site for the PA-2 prophage, we selected 20 independent PA-2 ts9 lysogens of strain EM3003 and used these as recipients in conjugation experiments. Strain CS411 was used as the donor with selection for aro+ recombinants in the presence of streptomycin (100 jig/ml) at 30°C as described by Miller (4). In each case, the majority of the recombinants were able to grow at 42°C indicating a high frequency of transfer of the ts9+ marker and close linkage between attPA-2 and aroC. In a further set of transduction experiments between CS396 and CS419, we also scored for transfer of the two other prophage markers, h (host range) and lc. The host range (h IPA-2 or h") of the progeny phage from the selected aroC+ recombinants was determined by assaying their ability to form plaques on strains CS208 (a Par mutant lacking the PA-2 receptor [2, 5]) and poplO75 (a LamB mutant lacking the A receptor). Production of protein 2 was determined by polyacrylamide gel electrophoresis. For this purpose we employed a novel membrane fractionation procedure in which very small volumes of cells are used. Cells growing rapidly in 6 ml of TY medium (5) containing 500 liM MgCl2 at 30°C (5 x 10' cells per ml) were treated with 5 ig of polymyxin B per ml for 10 min followed by 0.5% sodium dodecyl sulfate until lysis was complete (15 to 30 min). The cell debris (membrane fraction) was then pelleted by centrifugation at 46,000 x g for 10 min at 10°C and suspended in sample buffer for electrophoresis using the Trisglycine-hydrochloride buffer system described previously (2). This procedure produced a membrane fraction which contained some proteins from both the inner and outer membranes. The major outer membrane proteins 3a and 3b were not present, and proteins la, lb, and 2 were clearly resolved. The results of the transduction analysis (Table 2) indicate that the prophage markers ts9, lc, and h are all closely linked and that the lc 808
NOTES
VOL. 29, 1979
protein 2 is unknown at the present time. A chromosomal gene linked to par also appears to affect production of protein 2 (2). However, there does not appear to be a separate PA-2 gene controlling the levels of proteins la and lb in PA-2 lysogens. We have examined wild-type cells lysogenized with several independently isolated hy7lc phage and find that these strains produce normal (wild-type) levels of proteins la and lb. This presumably indicates that it is competition for a limited number of common potential insertion sites that regulates the number of copies of each of these proteins that are inserted into the membrane, and that synthesis of all three proteins is regulated by a feedback mechanism. Although PA-2 and A appear to be closely related, we have thus far been unable to demonstrate PA-2-mediated specialized transduc-
may be closer to dsd than the other two markers. We are thus able to define a region between dsd and aroC within which attPA-2 is situated (Fig. 1). The role of the Ic gene in the production of TABLE 1. Strains of E. coli K-12 Strain no. W 1485F CS208
pI)op105
CS:396
EM:3003 CS419 CS411
Relevant characteristics
thi pao- 10 thi HfrG6 lamnB204 h7l17c lsogen of W1485F
aog amoC porF thi dsdc lay iPsL
809
Source and reference C.G.S.C'. (3) M. Hofnung T'his paper (5) E. McFall This paper This paper
PA-2 ts9 lxsogen of EM,3003 HfrKI,16 thi-I -el- I thyA2'4 no/A 13 di-zz I'A-2 " Coli (;enetic Stock Center, New Haven. Cotnn.
TABLE 2. Transduction data
CS;39i (hv7c35)
Donor markers Selectedrnsdce transduced
Selected marker (colonies tested)
Recipient
P1 grown on
purF (272)
CS419 (put-F asoC dsd
CS396
Marker
No. )
PA-2 ts9'
8 (3) 116 (43)
aroC'
PA-2Ws9)
dsd' PA-2 t.(s9'
aro(C (27)))
CS419
co-
Frequency of recombinant classes purF
PA-2 (s9
dsd
+
+ +
+ +
+
-
No.
O
49 (18) 95 (35) 7 (3)
purE'
dsd5('
+
I 6
2)
+
W1485F
CS396
EM3(X)3 (purF aroC d/sd)
piiF'
ato' (1(X))
CS419
L-
L dsd
a)o()' (30)1)
C
1
1
1 17
purFE (Isd'
36
T'A-2 ts9' T'A-2 hA PA-2 1e
18
36 1 1
aroC
tS9
-
_
-
-
95 119
1II) (3)
t.sd'
1
0.87 1.41
+
(56)3
>
20
12
I 0.60 ] 0.50 J
purF
Lysogen
. a *~~~~~
%lb
I% %%
I
%;4~.-I1I %46
Non- lysogen
did
aioC
,0.34 L
puirF 0.58 X
FIG. 1. Detail of the linkage map of Escherichia coli K-12 in the region purF-dsd showing the region occupied by the PA-2 prophage. The shaded area represents the E. coli chromosome, and the hatched area represents a region of either E. coli or PA-2 DNA. The clear area represents the region that must be occupied by PA-2 DNA. Figures are map distances (in minutes) of the E. coli K-12 chromosome (1).
810
NOTES
tion of the aroC gene under conditions that work well for A-mediated transduction of gal (4). This work was supported by Public Health Service grant GM-18006 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Bachmann, B. J., K. B. Low, and A. L. Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 2. Bassford, P. J., D. L. Diedrich, C. A. Schnaitman, and P. Reeves. 1977. Outer membrane proteins of Escherichia coli. VI. Protein alteration in bacteriophage-resistant mutants. J. Bacteriol. 131:608-622.
J. VIROL. 3. Davidson, N., and W. Szybalski. 1971. Physical and chemical characteristics of lambda DNA, p. 45-82. In A. D. Hershev (ed.). The bacteriophage lambda. Cold Spring lHarborI Laboratory, Cold Spring Harbor. N.Y. 4. Miller, J. H. 1972. Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 5. Pugsley, A. P., and C. A. Schnaitman. 1978. Outer membrane proteins of Escherichia coli. VII. Evidence that bacteriophage-directed protein 2 functions as a pore. J. Bacteriol. 133:1181-1189. 6. Schnaitman, C. A., D. Smith, and M. F. deSalas. 1975. Temperate bacteriophage that causes production of a new major outer membrane protein in Escherichia coli. J. Virol. 15:1121-1130.
