Vol. 140, No. 3
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 817-824
0021-9193/79/12-0817/08$02.00/0
Cloning and Physical Mapping of the dnaA Region of the Escherichia coli Chromosome TORU MIKI,t MINORU KIMURA, SOTA HIRAGA, TOSHIO NAGATA, AND TAKASHI YURA* Institute for Virus Research, Kyoto University, Kyoto 606, Japan Received for publication 18 July 1979
The dnaA gene of Escherichia coli K-12, supposedly present in the deoxyribonucleic acid (DNA) of specialized transducing phage A i2l dnaA-2, was cloned onto plasmid pBR322. The new plasmid was named pMCR501. Physical analyses of DNAs of A i2l dnaA-2 and pMCR501 revealed the following. The A i2' dnaA-2 DNA retained the AsrIX1-2 and ninR5 deletions and imm21 substitution which were originally present in the parental phage. This size reduction was compensated for by the insertion-substitution segment (tna-dnaA region) in A i2' dnaA2 DNA. The fractional size of this segment was approximately 7 megadaltons (Md), or 10 kilobases, which was found to be the sum of the tna insertion subsegment of ca. 3.5 Md and the dnaA substitution subsegment of ca. 3.5 Md. Phage P1-mediated transductional mapping between the dnaA46 and tna mutations gave a cotransduction frequency of 84%, corresponding to approximately 5 kilobases. Thus, it is strongly suggested that the dnaA gene resides in the A i2l dnaA-2 DNA. Cleavage mapping with the restriction endonuclease of pMCR501 DNA confirmed that it was constructed by excising a BamHI fragment of 4.29 Md, containing the 3.5-Md dnaA substitution segment, from the X i" dnaA-2 DNA, inserting it into the sole BamHI cleavage site on pBR322. The dnaA gene of Escherichia coli plays a role which is essential specifically for the E. coli chromosome to initiate a round of replication (11). Primarily because conditionally lethal, temperature-dependent dnaA mutants have been isolated (11, 17, 31), it is thought that the dnaA gene codes for a protein. In the framework of the replicon model (14), the dnaA "protein" may function as a regulator or "initiator" and act specifically at the "replicator" (the replication origin) of the E. coli chromosome. This notion has been supported by the phenomenon of "integrative suppression" (4, 21, 27), as well as by isolation of dnaA mutants which are normal at low temperatures but are initiation defective at high temperatures (11, 31). Recently a cold-sensitive dnaA mutant which overinitiates at low temperatures was isolated, suggesting a relationship between regulation of dnaA product synthesis and regulation of replication initiation (17). An autoregulation hypothesis for the dnaA function has also been fornulated (10). There is a hint that the dnaA protein might interact with DNA-dependent RNA polymerase
(2).
ticipation in the initiation of DNA replication remains unknown. It is thus desirable to identify the dnaA product, to characterize its biochemical properties, and to decipher the structure of the dnaA gene itself. As the first step toward these final goals, we have cloned the dnaA gene. We previously reported the isolation of strains of specialized transducing phage A i'l dnaA (23). In this paper we describe further cloning of the dnaA gene of A i2' dnaA into plasmid pBR322 and physical analyses of DNAs of A i2' dnaA and the plasmid. In the accompanying paper by Kimura et al. (18), we present studies on the proteins specified by A i2" dnaA and on the isolation and characterization of new amber mutations that are very close to the dnaA gene. (Part of this work was included in a thesis by T. Miki as a partial fulfilment for a Ph.D. degree, submitted to Kyoto University, Kyoto, Japan, 1978, and was presented in the Annual Report of the Institute for Virus Research, Kyoto University [vol. 21, p. 1-26, 1978].) MATERIALS AND METHODS Bacterial, phage, and plasmid strains. The bacterial strains used were derivatives of E. coli K-12 and are listed in Table 1. Phage A i2' tna (6) and plasmid pBR322 (5) were generously supplied by W. J. Brammer and H. W. Boyer, respectively. Phage P1 vir,
In spite of the information that has been accumulated, the exact mechanism of dnaA part Present address: Department of Biochemistry, Yamaguchi University School of Medicine, Ube 755, Japan.
817
818
MIKI ET AL.
J. BACTERIOL.
TABLE 1. Bacterial strains used Strain Relevant genotypea Source (reference) AT2243-11 pyrE41 uhp-2 B. J. Bachmann BC73 tna phoS ilv C. Wada C600(A i2' ninR5) tonA21 (A i2' ninR5) This work C600SAr lam This work dnaA46 ilv CSH42 J. H. Miller (24) KH692 pyrE41 uhp-2 bglR bglB dnaA46 tna T. Horiuchi KH693 dnaA46 tna bglR bglB metE (23) KH697 dnaA46 recAl T. Horiuchi KH712 bglR 4gal-attA-bio (23) KH715 tonA21 (Ah) T. Horiuchi KY7126 bglR Agal-attA-bio (A cI857 S7::bglB) (A cI h') (23) KY7231 trpB9578 tna-2 rpsL recAl This work KY7243 trpB9578 tna-2 rpsL (A i2' tna::tna) (23) SP3b tna bglR bglB B. J. Bachmann W3350 Prototroph Laboratory stock Ymel(A- P2+) tyrT (P2) M. Imai a For gene symbols, see Bachmann et al. (1). "::" indicates that the phage genome is inserted in the bacterial gene or in its vicinity; "A" designates deletion; "( )" indicates that the phage is present as a prophage. b This strain carries a tna mutation, in contrast to the original description.
Apapa, A c1857 S7, A cI b2, A i2' c, and 480 were our laboratory stocks. Media and methods for marker identification. The minimal agar medium used was described previously (23). L broth medium (20) was used for transduction with P1 vir (13) and for culturing bacterial strains lysogenic for A or harboring a plasmid. The genetic markerspyrE, uhp, andphoS were checked by the methods of Schaefler and Maas (29), Kadner (15), and Echols et al. (9), respectively. The other markers were checked as described previously (23). Buoyant density analysis of phage particles. Phage suspensions, each of which contained ca. 1.5 x 107 plaque-forming units of A i2' dnaA-2, Apapa, A cI b2, or 480, were mixed to obtain a total volume of 1.3 ml, into which a 1.7-ml CsCl solution, saturated at 40C, was added. This mixture was centrifuged in a Beckman L2-65B ultracentrifuge with a SW50.1 rotor at 23,000 rpm for 20 h. The bottom of the tube was punctured and each drop was collected. Titers of Apapa (turbid plaque) and A cI b2 (clear plaque) were assayed on C600(A i2' ninR5). Those of )80 and A i2' dnaA-2 were assayed on C600SAr and KH715, respectively. Apapa (1.508 g/cm3), 480 (1.494 g/cm3), and A cI b2 (1.491 g/cm3) served as buoyant density references (3, 12). Electron microscopy. The heteroduplex analysis of phage DNA by electron microscopy was as described previously (23). Purification of plasmid and phage DNA. When a culture of plasmid-bearing cells was in mid-exponential phase (ca. 2 x 108 cells per ml), chloramphenicol was added to a final concentration of 180 pg/mi. After subsequent overnight incubation at 37°C, the cells were harvested by centrifugation. A crude plasmid DNA solution was prepared, on an enlarged scale, by the method of Meyers et al. (22), and the convalently closed circular DNA was purified from the crude extract by ethidium bromide-cesium chloride equilibrium centrifugatioiL (28). After the ethidium bromide was removed by isopropanol treatment, DNA was
dialyzed against DNA buffer (10 mM Tris-hydrochloride [pH 8.0], 1 mM EDTA, and 10 mM NaCl). Phage DNA was prepared as described previously (23). Enzymes. Restriction endonucleases EcoRI, HindIII, BamHI, and T4 DNA ligase were purchased from Miles Laboratories, Inc., PstI was from Boehringer Mannheim Corp., and AvaI was from Bethesda Research Laboratories. Restriction endonuclease digestion. DNA was digested with EcoRI, HindIII, and BamHI in a buffer containing 90 mM Tris-hydrochloride (pH 7.9) and 10 mM MgCl2. PstI and AvaI digestions were carried out in a buffer containing 90 mM Tris-hydrochloride (pH 7.5) and 10 mM MgCl2 and in a buffer containing 80 mM Tris-hydrochloride (pH 7.4), 10 mM MgC12, 30 mM NaCl, 0.2 mM EDTA, and 50 pg of bovine serum albumin per ml, respectively. Agarose gel electrophoresis was as described previously (23). ligation and transformation. Approximately 1.5 pg of restriction endonuclease-digested DNA was incubated with 0.7 U of T4 DNA ligase at 40C for 20 h in a ligation buffer containing 90 mM Tris-hydrochloride (pH 7.9), 60 mM MgCl2, 100 mM dithiothreitol, and 0.5 mM ATP. Bacteria were transformed by the published method (25). RESULTS Transductional mapping of genes near dnaA. We performed phage Pl-mediated transduction with eight markers, including dnaA, to determine their map positions on the chromosome (Table 2; Fig. 1). (Some of these data were briefly reported previously [23].) We found that the tna gene was much closer to dnaA (Table 2, experiments 1, 2, and 4) than the position allocated on the standard map (1). This was later confirmed by physical mapping (see below). We also found that the phoS gene was located clockwise to bglB (Table 2, experiment 3), opposite
CLONING AND PHYSICAL MAPPING OF dnaA
VOL. 140, 1979
819
TABLE 2. Transductional mapping of the markers around the dnaA gene Expt
1
2
3
4
Donor/recipient
AT2243-11/KH693
W3350/KH692
SP3/BC73
SP3/CSH42
Selected marker (no. of colonies tested)
uhp
dnaA+ (73)
Oa
0
0
lb
1
0 1 1 1
1 1 1 0
pyrE+ (103)
Unselected marker: dnaA
tna
Fre-
bgMR phoS
0 0 1 0
56 33 5 5 1
0
0
0
0
0 1 1 1
0 0 1 0
0 0 0 0
0 0 0 0
0 0 0 1 0
0 0 1 1 1
0 0 1 1 1 1 0
0 1 1 1 1 1 0
0 0 0 1 1 0 1
(%)
15 51 11 3 19 1
1 1 1 0
0 0 0 0 1 1 1
quency
0 1 0 0 1 0
0
ilv+ (100)
ilv+ (80)
bglB 0
0 1 1 1
58 5 7 29
0
1
70 6 13 1
8 1 1
5
KH712/BC73
ilv+ (296)
0 0 1 1
0 1 1 0
46 36 18 0
6
KY7126/BC73
ilv+ (104)
0 0 1 1
0 1 1 0
57 43 0 0
a 0, Marker from the recipient strain. b 1, Marker from the donor.
the locus given on the standard map (1). This conclusion was further supported by the cross using KY7126 as a donor, whose bglB gene was cut by integration of the A genome as a prophage therein; the cotransduction frequency between ilv and phoS was not affected by the presence of the prophage, whereas that between ilv and tna was (cf. experiments 5 and 6, Table 2). The other markers were found at positions consistent with the standard map. Isolation of A i' dnaA The procedure for isolating strains of specialized transducing phage A i1" dnaA was described previously (23). Briefly, specialized transducing phage A i2' tna (6) was integrated in the tna region of the E. coli chromosome, and the resulting lysogen (KY7243) was induced by UV irradiation to yield a lysate from which dnaA-transducing phage strains
were isolated. They were plaque-forming and retained the activity of transducing tna. One of the strains, A i2' dnaA-2, was further characterized. It forms plaques efficiently on a recA E. coli strain, KY7231 (thus Fec+), but does not form plaques on a P2 lysogen, Ymel(A-, P2+) (thus Spi+). It forms clearer plaques than the A i" tna parent, suggesting reduced ability for lysogenization. The genome of A i2' dnaA-2 therefore seems to contain an intact red gene, but attPOP' or int, or both, may be affected. The buoyant density of the A i2' dnaA-2 particle was determined to be 1.512 g/cm3. By using the equation formulated by Bellett et al. (3), we calculated that A i2' dnaA-2 DNA was 3.4% longer than Apapa DNA. Heteroduplex analysis of X in dnaA-2 DNA. Heteroduplex molecules formed between
820
MIKI ET AL.
J. BACTERIOL.
uncR,A
koriC
bg!IbglC Iasn dnaA I CrbsK tna ilv (O's
pyrE uhp
18i.5
81
1182S
82
8b
,I I, 1
I I I I
1170
1
1 11 1 11I
11
70 r1
b
54
H
11 11 l-r11I
23
*
11
I -I 1 11
33
01126
1
I1
11 11e
#4 ly
1I 11
11
1110
'1 I~S ( 0)
11
11 11
I
11
11
11
11
FIG. 1. Transductional mapping of the genes around dnaA with phage P1. The number above an arrow represents the cotransduction frequency (percent) between the two markers and was taken from the data in Table 2. A selected marker is shown at the tail of an arrow, and an unselected marker is shown at the head. Positions of uncA, uncB, oriC, and asn were reported previously (16, 23). The scale shows part of the 100-min standard map of E. coli K12 (1).
DNAs of A i2' dnaA-2 and A c1857 S7 were analyzed by electron microscopy (Fig. 2). It was revealed that A i2' dnaA-2 retained the ninR5 deletion (segment H, Fig. 2) and the deletion (segment B) between EcoRI cleavage sites 1 and 2 (AsrIAl-2); these two deletions were originally present in phage 540 of Murray and Murray (26). The parent of A i2 dnaA-2, A i2' tna, was constructed by inserting the E. coli chromosomal fragment into the sole HindIII cleavage site (56.4% position, Fig. 2) of phage 540 DNA (6). A i2' dnaA-2 DNA contained a bacterial segment which was about 22% of the A DNA unit in length. This segment replaced the A DNA, which was only 1% long (segment D). The origin of this asymmetric substitution can be explained by the following mechanism. Namely, A i2' tna was integrated into the tna region of the E. coli chromosome by reciprocal recombination using nucleotide sequence homology; a rare event occurred during the subsequent excision that connected att (or the region nearby) and a site further away from the left-side tna region, producing A i2l dnaA-2. The orientation of the integrated genome was such that subsequent induction produced plaque-forming
strains of X i dnaA. This mechanism and the structure of the A i2' dnaA-2 DNA were confirmed by additional electron microscopic analyses of heteroduplex molecules formed between DNAs of A cI857 S7 and A i2' tna, A i2' c and X iA tna, and A i' tna and A i2' dnaA-2 (data not shown). These analyses showed that the tna region, originally present as an insertion in A i2' tna, was 11.0% long (standard deviation, 0.4) and that the newly added dnaA region, as a substitution, was 11.2% long (standard deviation, 0.3). The 22% long segment of A i2l dnaA-2 DNA (Fig. 2) is thus the sum of the tna insertion (the left half) and dnaA substitution (the right half). These values and positions agree well with the results obtained by cleavage mapping using restriction endonucleases (see below). The distance between the dnaA and tna genes is thus less than 22.3% of the A DNA unit, or ca. 6.9 megadaltons (Md) corresponding to 10.5 kilobases, confirming the result of Pl-mediated transductional mapping (Table 2, Fig. 1; see Discussion). Cloning of the dnaA region into pBR322. The dnaA region of the A i21 dnaA-2 genome was further cloned into plasmid pBR322, which carries the genes for ampicillin resistance (Apr) and tetracycline resistance (Tcr). Five restriction endonucleases (EcoRI, HindIII, BamHI, PstI, and SalI), each of which cleaves at a single site on pRB322, were used, because cleavage and inactivation of the functional dnaA gene had to be avoided, but no information was available as to whether any of the enzymes could cut it. Thus, a mixture of pBR322 and A i2 dnaA-2 DNA was digested with each of the nucleases and then sealed with DNA ligase. The ligant was used to transform KH697 (dnaA46 recAl), with selection for temperature-resistant (growing at 4200) clones which were at the same time Apr or Tcr or Apr Tcr. Two plasmid clones, pMCR501 (constructed with BamHI) and pMCR511 (constructed with PstI), were thus obtained and subjected to cleavage mapping
analysis. Cleavage mapping of the dnaA region. Figure 3 illustrates the size and order of DNA fragments generated by digestion of purified DNAs from A i2l dnaA-2, A i2l tna, and pMCR501, using EcoRI, HindIII, BamHI and combinations of two. Size was estimated by agarose gel electrophoresis. The smallest HindIII fragment (3.7 Md) of A i2' tna DNA ("tna fragment") was isolated from the gel and further digested with EcoRI, BamHI, and EcoRI plus BamHI. This fragment corresponds to the tna subsegment of 11.0% A DNA unit (ca. 3.4 Md) shown in Fig. 2 and was inserted in the sole
CLONING AND PHYSICAL MAPPING OF dnaA
VOL. 140, 1979
6.25 (0. 18)
tO 0.05)
2.95 10.06)
43.29 (055)
10.01(0.19)
404
(0.02)
13.65(0.18)
_R _ ___1
-_
821
-__ 2Z28 (0.43)
_.
AC1857.)7 A
i2rdnaA-2
3.65 (0.08)
11.00 (0.42) 11.21 (0.31)
tna
dnaA
imm21
FIG. 2. Electron microscopic analysis ofheteroduplex DNA from A cI857 S7 and A i2l dnaA-2. Methods for the analysis were described previously (23). The bar on the electron micrograph represents 1 pm. The marker positions on the scale were assigned according to the literature: EcoRI-1, EcoRI-2, and HindIII (26); att (19); the endpoints of immA; and the endpoints of ninR5 (7). The numbers represent percent A DNA unit. The fractional length of each segment was estimated by taking the "boxed" numbers (8.6 for single-stranded segments and 10.4 for double-stranded segments) as references. Twenty heteroduplex molecules were analyzed, and standard deviations (numbers in parentheses) were calculated. The assignment of the tna and dnaA subsegments of the 22.3% long segment and estimation of their fractional length were carried out by additional analyses (see text).
HindJII site on phage 540 DNA, as noted before. structed by systematically comparing the two Thus, it served as the marker for construction of sets of fragments. The order gf the two EcoRI the cleavage map of X i2' tna DNA (Fig. 3). The fragments (2.1 and 0.63 Md) generated from A cleavage pattern of this fragment is completely i21 dnaA-2 DNA cannot primarily be determined different from that of pMCR501 DNA, clearly distinguishing the dnaA fragment from the tna fragment (Fig. 3). The order of EcoRI fragments for A i" tna (Fig. 3) was determined by using the map of A cI857 DNA (30) and the genetic structure of phage 540 (26) as references. Once the map of A i" tna DNA was established, the map of A i21 dnaA-2 DNA was con-
by this experiment alone, but molecular size estimation of some intermediates produced by partial digestion with EcoRI (data not shown) suggested the order shown in Fig. 3. This conclusion was supported by analysis of deletion derivatives of A i2l dnaA-2 (M. Kimura, unpublished data). Thus, pMCR501 contains the region whose cleavage pattern is identical to that
822
MIKI ET AL.
J. BACTERIOL.
O
10
5
iumVW v Rs v
A
n
A
0
a
"
a
c-
-NA
30 Md
l
I-
v
I
X
20
15
afi J4rJIl-2 i v
E G
4
I
WV
v
V.
&AO
v
A
.Atna
4
-!.= i.dnaA-2
pMCR501
dwA
tna 7.16
13.7
1.65
2.9
2.13
EcoRI v 13.36
3.51
14.47
05
Q94
3.7
a96
9A5
13.7
3.51
Hindill
2.96 165 2J3 48 083 2.1 2. 4.29 6.96 1W3 05 1~~~~I ~ ~~~
14.47 ~LI 37 '
12*
IP.I 1-~~
_AA
o
BamHI
HindUI
FIG. 3. Cleavage maps of A i2" tna, A i21 dnaA-2, and pMCR501 DNAs. The length of the wild-type A DNA is shown at the top. The open bar represents phage or plasmid DNA, the solid bar represents bacterial DNA, and the shaded area represents uncertainty. The molecular sizes (megadaltons) ofDNA fragments of A i2' tna and A i2l dnaA-2 produced by digestion with each enzyme are shown at the bottom. AsrIX1-2 represents a deletion of the EcoRI 3.0-Md fragment that was carried over from phage 540 ofMurray and Murray (26). Sites cleaved by EcoRI (V), HindIII (O), BamHI (A), and PstI (*) are indicated.
of the region in the A i2' dnaA-2 DNA that is adjacent to the tna region. It is concluded that pMCR501 indeed was produced by cleaving two BamHI sites on the A 1' dnaA-2 DNA (one near the HindIII site and the other near att), excising the 4.29-Md fragment containing the dnaA region, and inserting it into the BamHI site on pBR322. In Fig. 3, the PstI cleavage sites on pMCR501 are also indicated. Preliminary data for plasmid pMCR511 (constructed with PstI) showed that neither of the two PstI fragments generated from pMCR511 DNA corresponded to any of the PstI fragments from A i2 dnaA-2 DNA, but double digestions with PstI plus EcoRI, BamHI, or HindIII revealed that some fragments were similar, and the structure of the region around the EcoRI fragment of 0.63 Md on pMCR511 was similar to the region on pMCR501 and A i2l dnaA-2 DNAs (data not shown). The PstI fragment containing the dnaA region, cloned on pMCR511, might have been derived from a A i2' dnaA-2 variant with a spontaneous insertion or deletion. Alternatively, pMCR511 was possibly derived by spontaneous deletion from a larger plasmid which contained the intact PstI fragment. In this connection it may be noteworthy that pMCR501 was found to be somewhat unstable in maintaining its intact structure, and deletion derivatives could easily be isolated from it (T. Miki, unpublished data). In any event, comparison of the fragments produced by digestion of pMCR501 with PstI plus EcoRI, HindIII, or BamHI, and the result of digestion of the
"tna fragment" with PstI, allowed assignment of the PstI sites on pMCR501 as shown in Fig. 3.
The size of the dnaA region in the A i2' dnaA2 DNA can be estimated from the cleavage map to be ca. 3.6 Md; this region corresponds to the dnaA subsegment of 11.2% A DNA unit (ca. 3.5 Md) shown in Fig. 2.
DISCUSSION The DNA structure of specialized transducing phage A i2' dnaA-2 was determined by electron microscopic analyses of heteroduplex molecules and by cleavage mapping with restriction endonucleases. The sum of fractional lengths of the A i" dnaA-2 DNA segments estimated by electron microscopy (Fig. 2) indicated that the total length was 101.5% of the A DNA unit. The buoyant density of the A i21 dnaA-2 particles was 1.512 g/cm3, indicating that the DNA molecule is 3.4% longer than the Apapa DNA. The sum of fractional lengths of the A i2' dnaA-2 DNA fragments estimated by cleavage mapping (Fig. 3) was 100.9% of the A c1857 S7 DNA unit. These data agree well with each other and show that A i2' dnaA-2 DNA is as large as, or slightly larger than, A DNA. This indicates that the insertionsubstitution of the bacterial chromosome segment (the tna-dnaA region) compensated for the size reduction caused by the AsrIAl-2 and ninR5 deletions and by the imm21 substitution in the original phage no. 540 of Murray and Murray (26).
VOL. 140, 1979
CLONING AND PHYSICAL MAPPING OF dnaA
The size of the tna insertion segment estimated by electron microscopy was 11.0% of the X DNA unit; this corresponds to 3.4 Md when the molecular weight of A DNA is taken as 3.1 x 107 (8). The size of the same segment estimated by cleavage mapping was 3.7 Md. The size of the dnaA substitution segment estimated by electron microscopy was 11.2% of the A DNA unit, or 3.5 Md, and that by cleavage mapping was 3.6 Md. The overall tna-dnaA segment was estimated to be 22.3% of the A DNA unit; or 6.9 Md, by electron microscopy, and 7.3 Md by cleavage mapping. Thus, both of the methods gave consistent results which show that the dnaA region is located adjacent to the tna region on the E. coli chromosome and that the encompassing segment is integrated into A i2 dnaA-2 DNA. The distance between the dnaA46 and tna mutations was roughly estimated by phage P1mediated transductional mapping. The cotransduction frequency between the two markers was 84% (Table 2, experiment 1; Fig. 1). By using the Wu equation (32), we calculated that the distance between the two markers was 0.11 min (when the P1 transducing fragment was taken as equivalent to 2.0 min) or 0.13 min (when the P1 fragment was taken as 2.3 min). A molecular length of 1 min of the E. coli K-12 genetic map unit is now estimated to correspond to ca. 41 kilobases (1). Thus, the dnaA46 and tna mutations may be considered to be roughly 5 kilobases apart. This is clearly within the stretch of the insertion-substitution segment on the dnaA-2 DNA. The size of this segment
A
i2l
was cor-
approximately 7 Md, as noted above, which responds to ca. 10 kilobases. Although we cannot entirely exclude the possibility that the dnaAtransducing activity of A ei dnaA-2 might be due to some sort of suppression of the DnaAphenotype, without the presence and participation of the dnaA gene on the phage genome, the probability that the 10-kilobase segment contains the dnaA gene is very high. Since dnaAtransducing activity is expressed in cells lysogenic for A i2l dnaA-2, the dnaA gene, which is supposed to reside on the phage genome, is not under the phage repressor control, implying that the gene is endowed with its own promoter. From A il dnaA-2 DNA the segment including the dnaA substitution was further cloned into plasmid pBR322, and chimeric plasmids pMCR501 and pMCR511 were obtained. Cleavage mapping of pMCR501, which was constructed with BamHI, revealed that it acquired a BamHI fragment of 4.29 Md. Comparison of the map of this fragment with the maps of A il tna and A i2l dnaA-2 DNAs clearly shows that
823
the fragment was derived from the region including the dnaA substitution present on the parental A i21 dnaA-2 DNA (Fig. 3). Thus plasmid pMCR501 can be considered to carry the dnaA gene. It may be transcribed from its own promoter, although its presence on pMCR501 has not been studied, or from the promoter of the tetracycline resistance gene on the pBR322. Plasmids such as pMCR501 and pMCR511 will be useful for analysis of the dnaA product protein, of interaction between the protein and the chromosome replication origin (oriC) (23), and of the nucleotide sequence of the dnaA gene. ACKNOWLEDGMENTS We are grateful to B. J. Bachmann, H. W. Boyer, W. J. Brammer, T. Horiuchi, M. Imai, and C. Wada for bacterial, phage, and plasmid strains. We thank J. Asano and A. Komori for their expert technical assistance. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan. 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. Bagdasarian, M. M., M. Izakowska, and M. Bagdasarian. 1977. Suppression of the DnaA phenotype by mutations in the rpoB cistron of ribonucleic acid polymerase in Salmonella typhimurium and Escherichia coli. J. Bacteriol. 130:577-582. 3. Bellett, A. J. D., H. G. Busse, and R. L Baldwin. 1971. Tandem genetic duplication in a derivative of phage lambda, p. 501-513. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 4. Bird, R. E., M. Chandler, and L. Caro. 1976. Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: change of chromosome replication origin in synchronized cultures. J. Bacteriol. 126:1215-1223. 5. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, and H. W. Boyer. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. 6. Borck, K., J. D. Brammer, W. J. Hopkins, and N. E. Murray. 1976. The construction in vitro of transducing derivatives of phage lambda. Mol. Gen. Genet. 146: 199-207. 7. Chow, L. T., N. Davidson, and D. Berg. 1974. Electron microscope study of the structures of Adv DNAs. J. Mol. Biol. 86:69-89. 8. Davidson, N., and W. Szybalski. 1971. Physical and chemical characteristics of lambda DNA, p. 45-82. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 9. Echols, H., A. Garen, S. Garen, and A. Torriani. 1961. Genetic control of repression of alkaline phosphatase in E. coli. J. Mol. Biol. 3:425-438. 10. Hansen, F. G., and K. V. Rasmussen. 1977. Regulation of the dnaA product in Escherichia coli. Mol. Gen. Genet. 155:219-225. 11. Hirota, Y., J. Mordoh, and F. Jacob. 1970. On the process of cellular division in Escherichia coli. III. Thermosensitive mutants of Escherichia coli altered in the process of DNA initiation. J. Mol. Biol. 53:369-387. 12. Igarashi, K., S. Hiraga, and T. Yura. 1967. A deoxythynmidine kinase deficient mutant of Escherichia coli.
824
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MIKI ET AL.
II. Mapping and transduction studies with phage o80. Genetics 57:643-654. 13. Ikeda, H., and J. Tomizawa. 1965. Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragments. J. Mol. Biol. 14:85-109. 14. Jacob, F., S. Brenner, and F. Cuzin. 1963. On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28:329-348. 15. Kadner, R. J. 1973. Genetic control of the transport of hexose phosphates in Escherichia coli: mapping of the uhp locus. J. Bacteriol. 116:764-770. 16. Kanazawa, H., T. Mili, F. Tamura, T. Yura, and M. Futai. 1979. Specialized transducing phage A carrying the genes for coupling factor of oxidative phosphorylation of Escherichia coli: increased synthesis of coupling factor on induction of prophage Aasn. Proc. Natl. Acad. Sci. U.S.A. 76:1126-1130. 17. Kelienberger-Gujer, G., A. J. Podhajeka, and L. Caro. 1978. A cold sensitive dnaA mutant of E. coli which overinitiates chromosome replication at low temperature. Mol. Gen. Genet. 162:9-16. 18. Kimura, M., T. Miki, S. Hiraga, T. Nagata, and T. Yura. 1979. Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication. J. Bacteriol. 140: 825-834. 19. Landy, A., and W. Ross. 1977. Viral integration and excision: structure of the lambda att sites. Science 197: 1147-1160. 20. Lennox, E. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190206. 21. Lindahl, G., Y. Hirota, and F. Jacob. 1971. On the process of cellular division in Escherichia coli: replication of the bacterial chromosome under control of prophage P2. Proc. Natl. Acad. Sci. U.S.A. 68:2407-2411. 22. Meyers, J. A., D. Sanchez, L. P. Elwell, and S. Falkow. 1976. Simple agarose gel electrophoretic method
23.
24. 25. 26.
for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacteriol. 127:1529-1537. Miki, T., S. Hiraga, T. Nagata, and T. Yura. 1978. Bacteriophage A carrying the Escherichia coli chromosomal region of the replication origin. Proc. Natl. Acad. Sci. U.S.A. 75:5099-5103. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Molholt, B., and J. Doskoefl. 1978. Increased transformation frequency in E. coli. Biochem. Biophys. Res. Commun. 82:477-483. Murray, K., and N. E. Murray. 1975. Phage lambda receptor chromosome for DNA fragments made with
restriction endonuclease III of HaemphiUus influenzae
and restriction endonuclease I of Escherichia coli. J. Mol. Biol. 98:551-564. 27. Nishimura, Y., L. Caro, C. M. Berg, and Y. Hirota. 1971.-Chromosome replication in Escherichia coli. IV. Control of chromosome replication and cell division by an integrated episome. J. Mol. Biol. 55:441-456. 28. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dyebuoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:15141521. 29. Schaefler, S., and W. K. Maas. 1967. Inducible system for the utilization of ,B-glucosides in Escherichia coli. H. Description of mutant types and genetic analysis. J. Bacteriol. 93:264-272. 30. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91:315-328. 31. Wechsler, J. A., and J. D. Gross. 1971. Escherichia coli mutants temperature-sensitive for DNA synthesis. Mol. Gen. Genet. 113:273-284. 32. Wu, T. T. 1966. A model for three-point analysis of random general transduction. Genetics 64:405-410.
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 817-824
0021-9193/79/12-0817/08$02.00/0
Cloning and Physical Mapping of the dnaA Region of the Escherichia coli Chromosome TORU MIKI,t MINORU KIMURA, SOTA HIRAGA, TOSHIO NAGATA, AND TAKASHI YURA* Institute for Virus Research, Kyoto University, Kyoto 606, Japan Received for publication 18 July 1979
The dnaA gene of Escherichia coli K-12, supposedly present in the deoxyribonucleic acid (DNA) of specialized transducing phage A i2l dnaA-2, was cloned onto plasmid pBR322. The new plasmid was named pMCR501. Physical analyses of DNAs of A i2l dnaA-2 and pMCR501 revealed the following. The A i2' dnaA-2 DNA retained the AsrIX1-2 and ninR5 deletions and imm21 substitution which were originally present in the parental phage. This size reduction was compensated for by the insertion-substitution segment (tna-dnaA region) in A i2' dnaA2 DNA. The fractional size of this segment was approximately 7 megadaltons (Md), or 10 kilobases, which was found to be the sum of the tna insertion subsegment of ca. 3.5 Md and the dnaA substitution subsegment of ca. 3.5 Md. Phage P1-mediated transductional mapping between the dnaA46 and tna mutations gave a cotransduction frequency of 84%, corresponding to approximately 5 kilobases. Thus, it is strongly suggested that the dnaA gene resides in the A i2l dnaA-2 DNA. Cleavage mapping with the restriction endonuclease of pMCR501 DNA confirmed that it was constructed by excising a BamHI fragment of 4.29 Md, containing the 3.5-Md dnaA substitution segment, from the X i" dnaA-2 DNA, inserting it into the sole BamHI cleavage site on pBR322. The dnaA gene of Escherichia coli plays a role which is essential specifically for the E. coli chromosome to initiate a round of replication (11). Primarily because conditionally lethal, temperature-dependent dnaA mutants have been isolated (11, 17, 31), it is thought that the dnaA gene codes for a protein. In the framework of the replicon model (14), the dnaA "protein" may function as a regulator or "initiator" and act specifically at the "replicator" (the replication origin) of the E. coli chromosome. This notion has been supported by the phenomenon of "integrative suppression" (4, 21, 27), as well as by isolation of dnaA mutants which are normal at low temperatures but are initiation defective at high temperatures (11, 31). Recently a cold-sensitive dnaA mutant which overinitiates at low temperatures was isolated, suggesting a relationship between regulation of dnaA product synthesis and regulation of replication initiation (17). An autoregulation hypothesis for the dnaA function has also been fornulated (10). There is a hint that the dnaA protein might interact with DNA-dependent RNA polymerase
(2).
ticipation in the initiation of DNA replication remains unknown. It is thus desirable to identify the dnaA product, to characterize its biochemical properties, and to decipher the structure of the dnaA gene itself. As the first step toward these final goals, we have cloned the dnaA gene. We previously reported the isolation of strains of specialized transducing phage A i'l dnaA (23). In this paper we describe further cloning of the dnaA gene of A i2' dnaA into plasmid pBR322 and physical analyses of DNAs of A i2' dnaA and the plasmid. In the accompanying paper by Kimura et al. (18), we present studies on the proteins specified by A i2" dnaA and on the isolation and characterization of new amber mutations that are very close to the dnaA gene. (Part of this work was included in a thesis by T. Miki as a partial fulfilment for a Ph.D. degree, submitted to Kyoto University, Kyoto, Japan, 1978, and was presented in the Annual Report of the Institute for Virus Research, Kyoto University [vol. 21, p. 1-26, 1978].) MATERIALS AND METHODS Bacterial, phage, and plasmid strains. The bacterial strains used were derivatives of E. coli K-12 and are listed in Table 1. Phage A i2' tna (6) and plasmid pBR322 (5) were generously supplied by W. J. Brammer and H. W. Boyer, respectively. Phage P1 vir,
In spite of the information that has been accumulated, the exact mechanism of dnaA part Present address: Department of Biochemistry, Yamaguchi University School of Medicine, Ube 755, Japan.
817
818
MIKI ET AL.
J. BACTERIOL.
TABLE 1. Bacterial strains used Strain Relevant genotypea Source (reference) AT2243-11 pyrE41 uhp-2 B. J. Bachmann BC73 tna phoS ilv C. Wada C600(A i2' ninR5) tonA21 (A i2' ninR5) This work C600SAr lam This work dnaA46 ilv CSH42 J. H. Miller (24) KH692 pyrE41 uhp-2 bglR bglB dnaA46 tna T. Horiuchi KH693 dnaA46 tna bglR bglB metE (23) KH697 dnaA46 recAl T. Horiuchi KH712 bglR 4gal-attA-bio (23) KH715 tonA21 (Ah) T. Horiuchi KY7126 bglR Agal-attA-bio (A cI857 S7::bglB) (A cI h') (23) KY7231 trpB9578 tna-2 rpsL recAl This work KY7243 trpB9578 tna-2 rpsL (A i2' tna::tna) (23) SP3b tna bglR bglB B. J. Bachmann W3350 Prototroph Laboratory stock Ymel(A- P2+) tyrT (P2) M. Imai a For gene symbols, see Bachmann et al. (1). "::" indicates that the phage genome is inserted in the bacterial gene or in its vicinity; "A" designates deletion; "( )" indicates that the phage is present as a prophage. b This strain carries a tna mutation, in contrast to the original description.
Apapa, A c1857 S7, A cI b2, A i2' c, and 480 were our laboratory stocks. Media and methods for marker identification. The minimal agar medium used was described previously (23). L broth medium (20) was used for transduction with P1 vir (13) and for culturing bacterial strains lysogenic for A or harboring a plasmid. The genetic markerspyrE, uhp, andphoS were checked by the methods of Schaefler and Maas (29), Kadner (15), and Echols et al. (9), respectively. The other markers were checked as described previously (23). Buoyant density analysis of phage particles. Phage suspensions, each of which contained ca. 1.5 x 107 plaque-forming units of A i2' dnaA-2, Apapa, A cI b2, or 480, were mixed to obtain a total volume of 1.3 ml, into which a 1.7-ml CsCl solution, saturated at 40C, was added. This mixture was centrifuged in a Beckman L2-65B ultracentrifuge with a SW50.1 rotor at 23,000 rpm for 20 h. The bottom of the tube was punctured and each drop was collected. Titers of Apapa (turbid plaque) and A cI b2 (clear plaque) were assayed on C600(A i2' ninR5). Those of )80 and A i2' dnaA-2 were assayed on C600SAr and KH715, respectively. Apapa (1.508 g/cm3), 480 (1.494 g/cm3), and A cI b2 (1.491 g/cm3) served as buoyant density references (3, 12). Electron microscopy. The heteroduplex analysis of phage DNA by electron microscopy was as described previously (23). Purification of plasmid and phage DNA. When a culture of plasmid-bearing cells was in mid-exponential phase (ca. 2 x 108 cells per ml), chloramphenicol was added to a final concentration of 180 pg/mi. After subsequent overnight incubation at 37°C, the cells were harvested by centrifugation. A crude plasmid DNA solution was prepared, on an enlarged scale, by the method of Meyers et al. (22), and the convalently closed circular DNA was purified from the crude extract by ethidium bromide-cesium chloride equilibrium centrifugatioiL (28). After the ethidium bromide was removed by isopropanol treatment, DNA was
dialyzed against DNA buffer (10 mM Tris-hydrochloride [pH 8.0], 1 mM EDTA, and 10 mM NaCl). Phage DNA was prepared as described previously (23). Enzymes. Restriction endonucleases EcoRI, HindIII, BamHI, and T4 DNA ligase were purchased from Miles Laboratories, Inc., PstI was from Boehringer Mannheim Corp., and AvaI was from Bethesda Research Laboratories. Restriction endonuclease digestion. DNA was digested with EcoRI, HindIII, and BamHI in a buffer containing 90 mM Tris-hydrochloride (pH 7.9) and 10 mM MgCl2. PstI and AvaI digestions were carried out in a buffer containing 90 mM Tris-hydrochloride (pH 7.5) and 10 mM MgCl2 and in a buffer containing 80 mM Tris-hydrochloride (pH 7.4), 10 mM MgC12, 30 mM NaCl, 0.2 mM EDTA, and 50 pg of bovine serum albumin per ml, respectively. Agarose gel electrophoresis was as described previously (23). ligation and transformation. Approximately 1.5 pg of restriction endonuclease-digested DNA was incubated with 0.7 U of T4 DNA ligase at 40C for 20 h in a ligation buffer containing 90 mM Tris-hydrochloride (pH 7.9), 60 mM MgCl2, 100 mM dithiothreitol, and 0.5 mM ATP. Bacteria were transformed by the published method (25). RESULTS Transductional mapping of genes near dnaA. We performed phage Pl-mediated transduction with eight markers, including dnaA, to determine their map positions on the chromosome (Table 2; Fig. 1). (Some of these data were briefly reported previously [23].) We found that the tna gene was much closer to dnaA (Table 2, experiments 1, 2, and 4) than the position allocated on the standard map (1). This was later confirmed by physical mapping (see below). We also found that the phoS gene was located clockwise to bglB (Table 2, experiment 3), opposite
CLONING AND PHYSICAL MAPPING OF dnaA
VOL. 140, 1979
819
TABLE 2. Transductional mapping of the markers around the dnaA gene Expt
1
2
3
4
Donor/recipient
AT2243-11/KH693
W3350/KH692
SP3/BC73
SP3/CSH42
Selected marker (no. of colonies tested)
uhp
dnaA+ (73)
Oa
0
0
lb
1
0 1 1 1
1 1 1 0
pyrE+ (103)
Unselected marker: dnaA
tna
Fre-
bgMR phoS
0 0 1 0
56 33 5 5 1
0
0
0
0
0 1 1 1
0 0 1 0
0 0 0 0
0 0 0 0
0 0 0 1 0
0 0 1 1 1
0 0 1 1 1 1 0
0 1 1 1 1 1 0
0 0 0 1 1 0 1
(%)
15 51 11 3 19 1
1 1 1 0
0 0 0 0 1 1 1
quency
0 1 0 0 1 0
0
ilv+ (100)
ilv+ (80)
bglB 0
0 1 1 1
58 5 7 29
0
1
70 6 13 1
8 1 1
5
KH712/BC73
ilv+ (296)
0 0 1 1
0 1 1 0
46 36 18 0
6
KY7126/BC73
ilv+ (104)
0 0 1 1
0 1 1 0
57 43 0 0
a 0, Marker from the recipient strain. b 1, Marker from the donor.
the locus given on the standard map (1). This conclusion was further supported by the cross using KY7126 as a donor, whose bglB gene was cut by integration of the A genome as a prophage therein; the cotransduction frequency between ilv and phoS was not affected by the presence of the prophage, whereas that between ilv and tna was (cf. experiments 5 and 6, Table 2). The other markers were found at positions consistent with the standard map. Isolation of A i' dnaA The procedure for isolating strains of specialized transducing phage A i1" dnaA was described previously (23). Briefly, specialized transducing phage A i2' tna (6) was integrated in the tna region of the E. coli chromosome, and the resulting lysogen (KY7243) was induced by UV irradiation to yield a lysate from which dnaA-transducing phage strains
were isolated. They were plaque-forming and retained the activity of transducing tna. One of the strains, A i2' dnaA-2, was further characterized. It forms plaques efficiently on a recA E. coli strain, KY7231 (thus Fec+), but does not form plaques on a P2 lysogen, Ymel(A-, P2+) (thus Spi+). It forms clearer plaques than the A i" tna parent, suggesting reduced ability for lysogenization. The genome of A i2' dnaA-2 therefore seems to contain an intact red gene, but attPOP' or int, or both, may be affected. The buoyant density of the A i2' dnaA-2 particle was determined to be 1.512 g/cm3. By using the equation formulated by Bellett et al. (3), we calculated that A i2' dnaA-2 DNA was 3.4% longer than Apapa DNA. Heteroduplex analysis of X in dnaA-2 DNA. Heteroduplex molecules formed between
820
MIKI ET AL.
J. BACTERIOL.
uncR,A
koriC
bg!IbglC Iasn dnaA I CrbsK tna ilv (O's
pyrE uhp
18i.5
81
1182S
82
8b
,I I, 1
I I I I
1170
1
1 11 1 11I
11
70 r1
b
54
H
11 11 l-r11I
23
*
11
I -I 1 11
33
01126
1
I1
11 11e
#4 ly
1I 11
11
1110
'1 I~S ( 0)
11
11 11
I
11
11
11
11
FIG. 1. Transductional mapping of the genes around dnaA with phage P1. The number above an arrow represents the cotransduction frequency (percent) between the two markers and was taken from the data in Table 2. A selected marker is shown at the tail of an arrow, and an unselected marker is shown at the head. Positions of uncA, uncB, oriC, and asn were reported previously (16, 23). The scale shows part of the 100-min standard map of E. coli K12 (1).
DNAs of A i2' dnaA-2 and A c1857 S7 were analyzed by electron microscopy (Fig. 2). It was revealed that A i2' dnaA-2 retained the ninR5 deletion (segment H, Fig. 2) and the deletion (segment B) between EcoRI cleavage sites 1 and 2 (AsrIAl-2); these two deletions were originally present in phage 540 of Murray and Murray (26). The parent of A i2 dnaA-2, A i2' tna, was constructed by inserting the E. coli chromosomal fragment into the sole HindIII cleavage site (56.4% position, Fig. 2) of phage 540 DNA (6). A i2' dnaA-2 DNA contained a bacterial segment which was about 22% of the A DNA unit in length. This segment replaced the A DNA, which was only 1% long (segment D). The origin of this asymmetric substitution can be explained by the following mechanism. Namely, A i2' tna was integrated into the tna region of the E. coli chromosome by reciprocal recombination using nucleotide sequence homology; a rare event occurred during the subsequent excision that connected att (or the region nearby) and a site further away from the left-side tna region, producing A i2l dnaA-2. The orientation of the integrated genome was such that subsequent induction produced plaque-forming
strains of X i dnaA. This mechanism and the structure of the A i2' dnaA-2 DNA were confirmed by additional electron microscopic analyses of heteroduplex molecules formed between DNAs of A cI857 S7 and A i2' tna, A i2' c and X iA tna, and A i' tna and A i2' dnaA-2 (data not shown). These analyses showed that the tna region, originally present as an insertion in A i2' tna, was 11.0% long (standard deviation, 0.4) and that the newly added dnaA region, as a substitution, was 11.2% long (standard deviation, 0.3). The 22% long segment of A i2l dnaA-2 DNA (Fig. 2) is thus the sum of the tna insertion (the left half) and dnaA substitution (the right half). These values and positions agree well with the results obtained by cleavage mapping using restriction endonucleases (see below). The distance between the dnaA and tna genes is thus less than 22.3% of the A DNA unit, or ca. 6.9 megadaltons (Md) corresponding to 10.5 kilobases, confirming the result of Pl-mediated transductional mapping (Table 2, Fig. 1; see Discussion). Cloning of the dnaA region into pBR322. The dnaA region of the A i21 dnaA-2 genome was further cloned into plasmid pBR322, which carries the genes for ampicillin resistance (Apr) and tetracycline resistance (Tcr). Five restriction endonucleases (EcoRI, HindIII, BamHI, PstI, and SalI), each of which cleaves at a single site on pRB322, were used, because cleavage and inactivation of the functional dnaA gene had to be avoided, but no information was available as to whether any of the enzymes could cut it. Thus, a mixture of pBR322 and A i2 dnaA-2 DNA was digested with each of the nucleases and then sealed with DNA ligase. The ligant was used to transform KH697 (dnaA46 recAl), with selection for temperature-resistant (growing at 4200) clones which were at the same time Apr or Tcr or Apr Tcr. Two plasmid clones, pMCR501 (constructed with BamHI) and pMCR511 (constructed with PstI), were thus obtained and subjected to cleavage mapping
analysis. Cleavage mapping of the dnaA region. Figure 3 illustrates the size and order of DNA fragments generated by digestion of purified DNAs from A i2l dnaA-2, A i2l tna, and pMCR501, using EcoRI, HindIII, BamHI and combinations of two. Size was estimated by agarose gel electrophoresis. The smallest HindIII fragment (3.7 Md) of A i2' tna DNA ("tna fragment") was isolated from the gel and further digested with EcoRI, BamHI, and EcoRI plus BamHI. This fragment corresponds to the tna subsegment of 11.0% A DNA unit (ca. 3.4 Md) shown in Fig. 2 and was inserted in the sole
CLONING AND PHYSICAL MAPPING OF dnaA
VOL. 140, 1979
6.25 (0. 18)
tO 0.05)
2.95 10.06)
43.29 (055)
10.01(0.19)
404
(0.02)
13.65(0.18)
_R _ ___1
-_
821
-__ 2Z28 (0.43)
_.
AC1857.)7 A
i2rdnaA-2
3.65 (0.08)
11.00 (0.42) 11.21 (0.31)
tna
dnaA
imm21
FIG. 2. Electron microscopic analysis ofheteroduplex DNA from A cI857 S7 and A i2l dnaA-2. Methods for the analysis were described previously (23). The bar on the electron micrograph represents 1 pm. The marker positions on the scale were assigned according to the literature: EcoRI-1, EcoRI-2, and HindIII (26); att (19); the endpoints of immA; and the endpoints of ninR5 (7). The numbers represent percent A DNA unit. The fractional length of each segment was estimated by taking the "boxed" numbers (8.6 for single-stranded segments and 10.4 for double-stranded segments) as references. Twenty heteroduplex molecules were analyzed, and standard deviations (numbers in parentheses) were calculated. The assignment of the tna and dnaA subsegments of the 22.3% long segment and estimation of their fractional length were carried out by additional analyses (see text).
HindJII site on phage 540 DNA, as noted before. structed by systematically comparing the two Thus, it served as the marker for construction of sets of fragments. The order gf the two EcoRI the cleavage map of X i2' tna DNA (Fig. 3). The fragments (2.1 and 0.63 Md) generated from A cleavage pattern of this fragment is completely i21 dnaA-2 DNA cannot primarily be determined different from that of pMCR501 DNA, clearly distinguishing the dnaA fragment from the tna fragment (Fig. 3). The order of EcoRI fragments for A i" tna (Fig. 3) was determined by using the map of A cI857 DNA (30) and the genetic structure of phage 540 (26) as references. Once the map of A i" tna DNA was established, the map of A i21 dnaA-2 DNA was con-
by this experiment alone, but molecular size estimation of some intermediates produced by partial digestion with EcoRI (data not shown) suggested the order shown in Fig. 3. This conclusion was supported by analysis of deletion derivatives of A i2l dnaA-2 (M. Kimura, unpublished data). Thus, pMCR501 contains the region whose cleavage pattern is identical to that
822
MIKI ET AL.
J. BACTERIOL.
O
10
5
iumVW v Rs v
A
n
A
0
a
"
a
c-
-NA
30 Md
l
I-
v
I
X
20
15
afi J4rJIl-2 i v
E G
4
I
WV
v
V.
&AO
v
A
.Atna
4
-!.= i.dnaA-2
pMCR501
dwA
tna 7.16
13.7
1.65
2.9
2.13
EcoRI v 13.36
3.51
14.47
05
Q94
3.7
a96
9A5
13.7
3.51
Hindill
2.96 165 2J3 48 083 2.1 2. 4.29 6.96 1W3 05 1~~~~I ~ ~~~
14.47 ~LI 37 '
12*
IP.I 1-~~
_AA
o
BamHI
HindUI
FIG. 3. Cleavage maps of A i2" tna, A i21 dnaA-2, and pMCR501 DNAs. The length of the wild-type A DNA is shown at the top. The open bar represents phage or plasmid DNA, the solid bar represents bacterial DNA, and the shaded area represents uncertainty. The molecular sizes (megadaltons) ofDNA fragments of A i2' tna and A i2l dnaA-2 produced by digestion with each enzyme are shown at the bottom. AsrIX1-2 represents a deletion of the EcoRI 3.0-Md fragment that was carried over from phage 540 ofMurray and Murray (26). Sites cleaved by EcoRI (V), HindIII (O), BamHI (A), and PstI (*) are indicated.
of the region in the A i2' dnaA-2 DNA that is adjacent to the tna region. It is concluded that pMCR501 indeed was produced by cleaving two BamHI sites on the A 1' dnaA-2 DNA (one near the HindIII site and the other near att), excising the 4.29-Md fragment containing the dnaA region, and inserting it into the BamHI site on pBR322. In Fig. 3, the PstI cleavage sites on pMCR501 are also indicated. Preliminary data for plasmid pMCR511 (constructed with PstI) showed that neither of the two PstI fragments generated from pMCR511 DNA corresponded to any of the PstI fragments from A i2 dnaA-2 DNA, but double digestions with PstI plus EcoRI, BamHI, or HindIII revealed that some fragments were similar, and the structure of the region around the EcoRI fragment of 0.63 Md on pMCR511 was similar to the region on pMCR501 and A i2l dnaA-2 DNAs (data not shown). The PstI fragment containing the dnaA region, cloned on pMCR511, might have been derived from a A i2' dnaA-2 variant with a spontaneous insertion or deletion. Alternatively, pMCR511 was possibly derived by spontaneous deletion from a larger plasmid which contained the intact PstI fragment. In this connection it may be noteworthy that pMCR501 was found to be somewhat unstable in maintaining its intact structure, and deletion derivatives could easily be isolated from it (T. Miki, unpublished data). In any event, comparison of the fragments produced by digestion of pMCR501 with PstI plus EcoRI, HindIII, or BamHI, and the result of digestion of the
"tna fragment" with PstI, allowed assignment of the PstI sites on pMCR501 as shown in Fig. 3.
The size of the dnaA region in the A i2' dnaA2 DNA can be estimated from the cleavage map to be ca. 3.6 Md; this region corresponds to the dnaA subsegment of 11.2% A DNA unit (ca. 3.5 Md) shown in Fig. 2.
DISCUSSION The DNA structure of specialized transducing phage A i2' dnaA-2 was determined by electron microscopic analyses of heteroduplex molecules and by cleavage mapping with restriction endonucleases. The sum of fractional lengths of the A i" dnaA-2 DNA segments estimated by electron microscopy (Fig. 2) indicated that the total length was 101.5% of the A DNA unit. The buoyant density of the A i21 dnaA-2 particles was 1.512 g/cm3, indicating that the DNA molecule is 3.4% longer than the Apapa DNA. The sum of fractional lengths of the A i2' dnaA-2 DNA fragments estimated by cleavage mapping (Fig. 3) was 100.9% of the A c1857 S7 DNA unit. These data agree well with each other and show that A i2' dnaA-2 DNA is as large as, or slightly larger than, A DNA. This indicates that the insertionsubstitution of the bacterial chromosome segment (the tna-dnaA region) compensated for the size reduction caused by the AsrIAl-2 and ninR5 deletions and by the imm21 substitution in the original phage no. 540 of Murray and Murray (26).
VOL. 140, 1979
CLONING AND PHYSICAL MAPPING OF dnaA
The size of the tna insertion segment estimated by electron microscopy was 11.0% of the X DNA unit; this corresponds to 3.4 Md when the molecular weight of A DNA is taken as 3.1 x 107 (8). The size of the same segment estimated by cleavage mapping was 3.7 Md. The size of the dnaA substitution segment estimated by electron microscopy was 11.2% of the A DNA unit, or 3.5 Md, and that by cleavage mapping was 3.6 Md. The overall tna-dnaA segment was estimated to be 22.3% of the A DNA unit; or 6.9 Md, by electron microscopy, and 7.3 Md by cleavage mapping. Thus, both of the methods gave consistent results which show that the dnaA region is located adjacent to the tna region on the E. coli chromosome and that the encompassing segment is integrated into A i2 dnaA-2 DNA. The distance between the dnaA46 and tna mutations was roughly estimated by phage P1mediated transductional mapping. The cotransduction frequency between the two markers was 84% (Table 2, experiment 1; Fig. 1). By using the Wu equation (32), we calculated that the distance between the two markers was 0.11 min (when the P1 transducing fragment was taken as equivalent to 2.0 min) or 0.13 min (when the P1 fragment was taken as 2.3 min). A molecular length of 1 min of the E. coli K-12 genetic map unit is now estimated to correspond to ca. 41 kilobases (1). Thus, the dnaA46 and tna mutations may be considered to be roughly 5 kilobases apart. This is clearly within the stretch of the insertion-substitution segment on the dnaA-2 DNA. The size of this segment
A
i2l
was cor-
approximately 7 Md, as noted above, which responds to ca. 10 kilobases. Although we cannot entirely exclude the possibility that the dnaAtransducing activity of A ei dnaA-2 might be due to some sort of suppression of the DnaAphenotype, without the presence and participation of the dnaA gene on the phage genome, the probability that the 10-kilobase segment contains the dnaA gene is very high. Since dnaAtransducing activity is expressed in cells lysogenic for A i2l dnaA-2, the dnaA gene, which is supposed to reside on the phage genome, is not under the phage repressor control, implying that the gene is endowed with its own promoter. From A il dnaA-2 DNA the segment including the dnaA substitution was further cloned into plasmid pBR322, and chimeric plasmids pMCR501 and pMCR511 were obtained. Cleavage mapping of pMCR501, which was constructed with BamHI, revealed that it acquired a BamHI fragment of 4.29 Md. Comparison of the map of this fragment with the maps of A il tna and A i2l dnaA-2 DNAs clearly shows that
823
the fragment was derived from the region including the dnaA substitution present on the parental A i21 dnaA-2 DNA (Fig. 3). Thus plasmid pMCR501 can be considered to carry the dnaA gene. It may be transcribed from its own promoter, although its presence on pMCR501 has not been studied, or from the promoter of the tetracycline resistance gene on the pBR322. Plasmids such as pMCR501 and pMCR511 will be useful for analysis of the dnaA product protein, of interaction between the protein and the chromosome replication origin (oriC) (23), and of the nucleotide sequence of the dnaA gene. ACKNOWLEDGMENTS We are grateful to B. J. Bachmann, H. W. Boyer, W. J. Brammer, T. Horiuchi, M. Imai, and C. Wada for bacterial, phage, and plasmid strains. We thank J. Asano and A. Komori for their expert technical assistance. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan. 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. Bagdasarian, M. M., M. Izakowska, and M. Bagdasarian. 1977. Suppression of the DnaA phenotype by mutations in the rpoB cistron of ribonucleic acid polymerase in Salmonella typhimurium and Escherichia coli. J. Bacteriol. 130:577-582. 3. Bellett, A. J. D., H. G. Busse, and R. L Baldwin. 1971. Tandem genetic duplication in a derivative of phage lambda, p. 501-513. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 4. Bird, R. E., M. Chandler, and L. Caro. 1976. Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: change of chromosome replication origin in synchronized cultures. J. Bacteriol. 126:1215-1223. 5. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heyneker, and H. W. Boyer. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95-113. 6. Borck, K., J. D. Brammer, W. J. Hopkins, and N. E. Murray. 1976. The construction in vitro of transducing derivatives of phage lambda. Mol. Gen. Genet. 146: 199-207. 7. Chow, L. T., N. Davidson, and D. Berg. 1974. Electron microscope study of the structures of Adv DNAs. J. Mol. Biol. 86:69-89. 8. Davidson, N., and W. Szybalski. 1971. Physical and chemical characteristics of lambda DNA, p. 45-82. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 9. Echols, H., A. Garen, S. Garen, and A. Torriani. 1961. Genetic control of repression of alkaline phosphatase in E. coli. J. Mol. Biol. 3:425-438. 10. Hansen, F. G., and K. V. Rasmussen. 1977. Regulation of the dnaA product in Escherichia coli. Mol. Gen. Genet. 155:219-225. 11. Hirota, Y., J. Mordoh, and F. Jacob. 1970. On the process of cellular division in Escherichia coli. III. Thermosensitive mutants of Escherichia coli altered in the process of DNA initiation. J. Mol. Biol. 53:369-387. 12. Igarashi, K., S. Hiraga, and T. Yura. 1967. A deoxythynmidine kinase deficient mutant of Escherichia coli.
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