Vol. 174, No. 2
JOURNAL OF BACTERIOLOGY, Jan. 1992, p. 398-407
0021-9193/92/020398-10$02.00/0 Copyright © 1992, American Society for Microbiology
Involvement of Fis Protein in Replication of the Escherichia coli Chromosome MARCIN FILUTOWICZ,* WILMA ROSS, JADWIGA WILD, AND RICHARD L. GOURSE Department of Bacteriology, E. B. Fred Hall, 1550 Linden Drive, University of
Wisconsin-Madison, Madison, Wisconsin 53706 Received 20 August 1991/Accepted 18 November 1991
We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in thesefis mutant strains following upshift to 44°C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32°C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.
The initiation of DNA replication in Escherichia coli is a complex process which must be carried out with high accuracy under the diverse environmental conditions encountered by a cell. The initiation of DNA replication is very sensitive to modest changes in DNA supercoiling in vitro (1). Changes in DNA structure in vivo are known to occur in response to variation in temperature (20, 70), osmolarity (23), nutrients (2), or availability of oxygen (9). Therefore, the cell must have a means of compensating for the effects of alterations in DNA structure in order to carry out initiation of replication under a range of conditions. A class of abundant low-molecular-mass DNA binding proteins plays an accessory role in a variety of complex processes in vivo, including DNA replication, recombination, and transcription (see references 10, 17, and 59 for reviews). A common feature of HU and integration host factor (IHF), two extensively studied examples of these proteins, is the ability to bend the DNA to which they bind (27, 50). IHF can bind and bend oriC DNA at a unique site (15, 49). In a fractionated in vitro replitation system, both HU and IHF either stimulate replication or inhibit it in a concentration-dependent fashion (1, 5, 7, 45). E. coli produces several other small and abundant DNA binding proteins in addition to HU and IHF (10, 17, 59). In this study, we investigated the possible role of one of these, the Fis protein, in DNA replication. Fis is a 12-kDa protein which binds to DNA in a sequence-specific manner (6, 21, 28, 31, 32, 34-36, 54, 66). Fis was discovered initially as a host factor required to carry out the phage Mu gin and Salmonella typhimurium hin site-specific DNA inversion reactions in vitro (31, 34). Fis also participates in a related recombination system, cin, of phage P1 (21) and stimulates the excisive recombination of phage X (66). Fis was recently shown to activate rRNA transcription both in vivo and in vitro via interactions with an upstream activator region (54). We present in this report several lines of evidence indicating that the Fis protein plays a role in initiation of replication of the E. coli chromosome.
MATERIALS AND METHODS Bacterial strains and plasmids. E. coli strains used in this
study included the following: CSH50 [ara A(pro-lac) rpsL thi] and CSH50 fis::kan (36); MC1000 [AlacX74 araD139 A(ara-leu)7697 galU galK strA]; RJ1617 (MC1000 fis: :kan767) from Reid Johnson; CAG4000 (MG1655 AlacX74) from C. Gross and D.-J. Jin; RLG1347 (CAG4000 fis::kan) (54); EC6 (argE thi ilv) and EC8 [argE thi dnaA46(Ts)] (12); EC1510 [argE thi gyrB4J(Ts)] and EC1512 [argE thi gyrB402(Ts)] (14); and SD108 (trpE pyrF gyrB*) (51). fis::kan derivatives of some of the above strains were constructed by P1 transduction with P1 vir grown on either CSH50 fis::kan or RJ1617, with selection for kanamycin resistance (50 ,ug/ml). Throughout this work, the fis allele from CSHSOfis: :kan will be referred to asfis: :kan, and thefis allele from RJ1617 will be referred to as fis767. The newly constructed fis mutant strains include the following: ECF5201 (EC6 fis::kan), ECF5202 (EC6 fis767), ECF5203 (EC1510fis767), ECF5204 (EC1512fis767), ECF5206 (SD108 fis767), and RLG1863 (CAG4000fis767). Plasmid pLAR7 contains both the pBR322 origin of replication and oriC (53), and the isogenic plasmid pKO6 contains only the pBR322 origin (53). Plasmid pOC15 contains oriC as its only origin of replication (68). Media and growth conditions. Bacteria were grown on Luria-Bertani (LB) agar without or, where indicated, with 1% glucose. M9 complete medium used in experiments measuring DNA synthesis was supplemented with 0.5% Casamino Acids (Difco), 2% glucose, 5 ,ug of thymidine (Sigma) per ml, 250 ,g of deoxyadenosine (Sigma) per ml, and [3H]thymidine (1.8 uCi/,Lg/ml; Amersham). Transformation efficiency and plasmid segregation. Competent cells and plasmid DNAs were prepared according to published procedures (40). Plasmid segregation tests were performed as described in footnote a to Table 2. Individual colonies were examined for plasmid retention by touching a colony edge with a toothpick and transferring the material to the selective media. Fluorescence microscopy. The fluo-phase combined method of Hiraga (24) was modified as follows. Cells from a plate were suspended in a drop of 0.9% NaCl on a glass slide. The sample was heat fixed, methanol washed for 5 min, washed with tap water for 5 s, and air dried for approxi-
* Corresponding author. 398
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mately 20 min. Slides were stained with 10 RI of a 2.5-,ug/ml solution of 4',6-diamidino-2-phenylindole (Sigma) in 0.9% NaCl. A Zeiss photomicroscope with a NeoFluar 100/1, 30
Oel objective was used. Kodak Ektachrome 400 film was used for photography. DNase "footprinting." Six-microgram aliquots of pLAR7 DNA, purified on a Qiagen column (Qiagen Inc.), were digested with either SalI or HindIII, phenol extracted and ethanol precipitated, and then labeled for 5 min at 30°C with Sequenase (U.S. Biochemicals) and either [a-32P]TTP in the presence of 0.75 mM dideoxy-CTP for the SalI digest or [a-32P]dATP and 0.75 mM dideoxy-GTP for the HindIII digest. Labeled DNA was recut with either XhoI or Sail, and the fragment containing oriC was gel purified. Binding reaction mixtures (25 ,l) contained approximately 4 ng of purified DNA fragment in a solution containing 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 150 mM NaCl (or 80 mM NaCl), 1 mM dithiothreitol, 100 ,ug of bovine serum albumin per ml, and the indicated concentrations of purified Fis or IHF. Freshly prepared dilutions of Fis or IHF were made in 25 mM Tris-HCI (pH 7.5)-10% glycerol-50 mM NaCl. DNase I (Worthington) was added to a final concentration of 1 to 4 ,ug/ml and incubated for 30 s at room temperature, and the reaction was terminated by rapid phenol extraction in the presence of 10 mM EDTA. Sonicated calf thymus DNA (2.5 Vug) was added as a carrier for ethanol precipitation. Conditions of gel electrophoresis and autoradiography were as described previously (54). RESULTS fis mutants form filamentous cells at 44°C with reduced numbers of nucleoids and aberrant nucleoid segregation. E. coli strains carrying fis mutations in which a kanamycin resistance cassette was inserted into the intact fis gene (fis::kan [36]) or into a partially deletedfis gene (fis767 [32]) grow well under standard laboratory conditions (32, 36). However, we found that these fis null mutants failed to form individual colonies at 44°C on LB agar plates or at 42°C on minimal glucose plates containing Casamino Acids although there was residual growth in areas of heavy inoculum. At permissive temperatures, fis mutant strains form colonies with distinctive morphology (wrinkled edges). This feature is characteristic of E. coli mutants with perturbed cell division
(8).
Cell shape and nucleoid distribution in fis mutant and wild-type cells were compared by using phase-fluorescence microscopy (Fig. 1). Wild-type cells are short and nucleated irrespective of the temperature of growth. In contrast,fis767 andfis::kan strains display pronounced filamentation at 44°C and a lesser, although still considerable, degree of filamentation at 37 or 32°C (Fig. 1 and data not shown). The localization of DNA as judged by 4',6-diamidino-2-phenylindole staining differs in fis mutant cells grown at different temperatures. Staining in cells incubated at 44°C is very intense and distributed unevenly in the filament. Some anucleate cells are observed at 44°C. In cells grown at 37°C, DNA staining is less intense and is distributed throughout the filament. The morphological and DNA staining characteristics of the fis mutant cells at high temperatures are reminiscent of strains deficient in DNA replication (3, 8, 67). Therefore, we measured DNA synthesis directly in fis mutants at different temperatures.
DNA synthesis is inhibited in fis mutants exposed to high temperatures. The kinetics of incorporation of [3H]thymidine in wild-type cells (Fig. 2A) was compared with that in fis
399
mutant cells (Fig. 2B) at 32°C and following a shift to 44°C (time = 0). Incorporation is comparable in wild-type and mutant cells at 32°C. However, following a shift to 44°C, DNA synthesis in the fis mutant cells stops after approximately four chromosome equivalents of DNA are made (Fig. 2B). Conditional missense mutants affecting different components of the DNA replication machinery synthesize various amounts of DNA prior to inhibition. Mutants affecting DNA elongation show an immediate arrest in synthesis upon shift to nonpermissive conditions, while mutants affecting the initiation stage synthesize on average a third of a chromosome equivalent before inhibition occurs (reference 42 and references therein). The long delay in inhibition observed in fis null mutants after shift to 44°C does not resemble either of these two patterns but does resemble the inhibition observed with conditional mutants in the ,B subunit of DNA gyrase or when inhibitors of the P subunit of gyrase are used (12-14, 47, 56, 62). However, it should be noted that thefis mutation used contains a deletion of much of the fis gene, and, therefore, it does not make a temperature-sensitive protein as do the gyrase mutants. Rather, the requirement of the cell for Fis is dependent upon temperature. Rifampin inhibits resumed DNA synthesis in fis mutants following temperature downshift, but chloramphenicol does not. After prolonged incubation at 44°C, followed by downshift to 32°C, DNA synthesis resumes in fis mutant cells without a lag and appears comparable to that in wild-type cells (Fig. 2A and B). Initiation of DNA replication in vivo requires a transcriptional step (37) and de novo protein synthesis (39). We asked, therefore, whether new RNA synthesis or new protein synthesis was required in order for DNA synthesis to resume upon downshift to 32°C. As expected, in wild-type cells, which contain initiation-proficient complexes at 44°C, DNA synthesis continues, presumably at already established replication forks, when further RNA or protein synthesis is blocked by rifampin or chloramphenicol prior to downshift (Fig. 2A). Synthesis of approximately one-third of a chromosome equivalent occurs in wild-type cells before rifampin or chloramphenicol inhibits new initiations. Infis mutant cells, chloramphenicol addition 5 min prior to temperature downshift does not block renewed DNA synthesis, and approximately one chromosome equivalent of DNA is synthesized before replication rounds terminate. Thus, no new protein synthesis is required for DNA synthesis to resume, and the synthesis of approximately one chromosome equivalent of DNA (rather than one-third as observed for the wild type) suggests that the chromosomes of fis mutant cells are synchronized by prolonged exposure to 44°C. In contrast, no resumption of DNA synthesis occurs if fis mutant cells are treated with rifampin before temperature downshift. We interpret these results to mean that the temperature shift is changing the requirements for replication and that Fis is required at or before the transcriptional step in initiation of replication at 44°C. Enhanced temperature sensitivity of DNA replication in gyrB fis double mutants. fis mutants and gyrB(Ts) mutants behave similarly in the DNA synthesis experiments described above (14, 47), suggesting that the loss of Fis and inactivation of gyrase might affect the same step(s) in initiation of replication. We utilized a genetic approach to ask whether DNA synthesis is more vulnerable to elevated temperature in double mutants carrying gyrB and fis::kan alleles. Isogenic double mutant strains were constructed by P1 transduction by using either of two fis::kan alleles and
400
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Wild-type
fis767
320C
370C
440C
FIG. 1. Phase-fluorescence micrographs of a wild-type strain (CAG4000) and the isogenicfis767 mutant (RLG1863) grown at the indicated temperatures on LB agar supplemented with 1% glucose. Although fis mutants do not form colonies at 440C, they exhibit residual growth in areas of heavy inoculation. Wild-type or fis mutant cells from areas of heavy inoculum were prepared for microscopy by using 4',6-diamidino-2-phenylindole staining as described in Materials and Methods. Nucleoids fluoresce brightly while cells scatter light under these conditions. All panels are at the same magnification (X2,000).
Fis PROTEIN IN E. COLI CHROMOSOME REPLICATION
VOL. 174, 1992
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B
A 100 0
a
S a a 0
- 10-
U
+
10
0
U1
a5i
.
E .
0
100 Tlme (min)
100
, 200
300
Time (min)
FIG. 2. The effect of temperature on DNA synthesis in a wild-type strain (EC6) (A) and the isogenicfis767 mutant strain (ECF5202) (B). Strains were grown at 32°C in complete M9 medium containing [3H]thymidine (as indicated in Materials and Methods). At a cell density of 107 per ml (time = 0), the culture was divided into two parts: one part was incubated further at 32°C (0), and the other was shifted to 44°C (@). At the time indicated (arrow), the cultures grown at 44°C were diluted fourfold in fresh medium prewarmed to 44°C, and the following antibiotics were added 5 min prior to the temperature downshift: none (A), 200 ,g of rifampin per ml (l), and 150 ,ug of chloramphenicol per ml (G). One-milliliter aliquots of the cultures were taken at the times indicated for determination of trichloroacetic acid-precipitable radioactivity. The optical density at 650 nm of cell cultures was monitored up to the point of temperature downshift, and no inhibition of cell growth was found for either wild-type orfis767 mutant cells (data not shown).
either of two temperature-sensitive gyrB alleles (gyrB4J or gyrB402) or the gyrB* allele. gyrB41 and gyrB402 are known to affect chromosomal supercoiling in a temperature-dependent fashion (64) and to inhibit initiation of DNA replication at nonpermissive temperatures (13, 14, 47). In addition, gyrB402 is believed to affect the elongation phase of DNA synthesis at 42°C (14). In the gyrB* mutant, overall supercoiling of the DNA is reduced by approximately 30% compared with that in wild-type cells (51). The isogenic sets of single and double mutant strains were tested for growth at different temperatures on LB plates (Fig. 3). Each of the double mutant strains shows a pattern of temperature sensitivity different from that of its fis mutant parent or its gyr mutant parent. The double mutant gyrB402 fis767 does not grow at 37°C, and the double mutant gyrB41 fis767 grows more slowly at 37°C, although temperatures above 42 or 44°C are required to prevent growth of the single mutant parent strains. The gyrB* mutation, which is not a temperature-sensitive gyrB allele, enhances the sensitivity of the fis767 mutant to elevated temperatures; the gyrB* fis767 double mutant grows poorly at 32 and 37°C and does not grow at 41°C. To determine whether the enhanced sensitivity to temper-
320C
ature in the gyrB fis double mutants is caused by the inhibition of DNA replication, the incorporation of [3H]thymidine into DNA was measured at 32°C and at 38°C in the single mutant strains (either gyrB41 or fis767) and in the double mutant gyrB41 fis767. In each of the single mutant strains, incorporation of [3H]thymidine increases exponentially with time at both temperatures (Fig. 4A and B). However, DNA synthesis is inhibited at 38°C in the gyrB41 fis767 double mutant (Fig. 4C), even though the optical density increases at this temperature (data not shown). Similar inhibition of DNA synthesis was observed with the gyrB402 fis767 double mutant strain, and the temperature required to inhibit synthesis was even lower (36°C). These results indicate that the gyrB(Ts) and the fis mutations synergistically affect DNA synthesis. DnaA protein is required for initiation of replication at oriC (26). Despite repeated attempts, we were unable to introduce the fis alleles into the EC8 [dnaA46(Ts)] strain by P1 transduction. It is important to point out, however, that this apparent incompatibility may be influenced by genetic background since a viable dnaA46fis767 double mutant has been constructed by another laboratory. However, the dou-
370 C
410 C
FIG. 3. The effect of temperature on growth of wild-type (WT) andfis and gyrB single mutant and double mutant strains. Bacteria from a single colony of the strains indicated were streaked onto LB agar plates and incubated at the indicated temperatures for 24 h.
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A
B
C
0
a
S1
U.
01
0
11
3.
C1
11
4-
I
100
200
100
0
200
100
0
Time (min)
Time (min)
Tinme (mln)
FIG. 4. The effect of temperature on DNA synthesis in isogenic strains gyrB41(Ts) (EC1510) (A), fis767 (ECF5202) (B), and gyrB41 fis767 (ECF5203) (C). The strains were grown at 30°C in complete M9 medium containing [3H]thymidine (as indicated in Materials and Methods), and at a cell density of 107 cells per ml, the culture was divided into two parts; one part was incubated further at 30°C (A), and the other one was shifted to 38°C (A). One-milliliter aliquots were taken at the times indicated for determination of trichloroacetic acid-precipitable radioactivity.
from the fis mutant strains is more frequent than from the isogenicfis' strains. At 30°C, pOC15 is lost two- to threefold more frequently from fis mutant than from fis+ strains. This loss is greater at high temperatures, with no detectable retention of pOC15 at 42°C in experiment A and five- to sixfold-reduced retention, compared with fis+ cells, at 42°C in experiment B. Thus, we conclude that in the absence of Fis protein, maintenance of oriC minichromosomes is further impaired, perhaps because of the direct involvement of Fis in replication. Fis protein binds to the minimal oriC. Since fis mutations affect initiation of replication, we tested whether Fis binds to the minimal oriC region in DNase I footprinting experiments. Such binding might influence oriC function by altering local DNA structure and/or by affecting interactions between oriC and other proteins. Purified Fis was found to protect two sites of relatively high affinity: Fis I, centered at position 202 and partially overlapping DnaA site R2, and Fis II, centered at approximately position 282, adjacent to DnaA site R4 (Fig. 5A to C and Fig. 6). In each of these sites, approximately 25 bp of sequence is protected. The protected regions are interrupted by two areas of enhanced cleavage (or lack of protection), separated by approximately 12 bp and offset on the two strands by 2 to 3 bp (Fig. 5A to C and Fig. 6). Positions of enhanced DNase I cleavage within Fis footprints have been described for other systems (6, 54) and are located in characteristic positions with respect to the degenerate Fis consensus sequence G/T--YR--A/T--YR--C/A (28). On the basis of the boundaries of protection at relatively low Fis concentrations and the location of enhanced or unprotected positions within Fis site I (Fig. 5A to C), we
ble mutant strain is more temperature sensitive than either the fis767 or the dnaA46 parent (30a). oriC minichromosomes are not stably maintained in fis mutants. The replication of oriC requires almost the same set of factors whether it is a part of the intact chromosome or present on an extrachromosomal plasmid (see reference 69 for a review). We asked, therefore, whether replication of oriC minichromosomes requires Fis protein. Plasmids carrying the pBR322 origin (pLAR7 and pKO6) transform each of the isogenic fis+, fis::kan, and fis767 strains used with a comparable frequency irrespective of temperature (Table 1). The oriC-dependent plasmid pOC15, however, transforms the fis mutant strains with significantly reduced efficiencies at 42°C compared with those at 30°C, while it transforms the wild-type (fis') strains with comparable efficiencies at the two temperatures. (Relatively low transformation efficiency of wild-type strains is a characteristic feature of oriC plasmids [68, 69].) The failure to transform fis mutant strains with pOC15 at 42°C is not caused by growth inhibition of recipient bacteria by extra copies of oriC and/or overreplication of oriC, since the plasmid containing both oriC and the pBR322 origin can be efficiently transformed under these conditions. The stability of afis mutant pOC15 transformant obtained at 30°C was then evaluated at 42°C by using two different plasmid segregation tests (Table 2). The control plasmids pLAR7 and pKO6 are stably maintained irrespective of host and temperature of incubation. The oriC plasmid pOC15, as previously shown (references 15 and 69 and references therein) segregates frequently in wild-type cells irrespective of temperature. However, the loss of the pOC15 plasmid
TABLE 1. Reduced transformation frequency of fis mutants with oriC plasmid pOC15 Recipient strain at tempa
fis+
Plasmid pKO6 pLAR7 pOC15
fis767
fis::kan
300C
42°C
300C
42°C
1.5 x 10-4 2.1 x 10-4 1.2 x 10-6
1.6 x 10-4 1.9 X 10-4 1.3 x 10-6
1.2 x 10-4 1.9 X 10-4 1.7 x 10-6
1.7 x 10-4 2.4 x 10-4
JOURNAL OF BACTERIOLOGY, Jan. 1992, p. 398-407
0021-9193/92/020398-10$02.00/0 Copyright © 1992, American Society for Microbiology
Involvement of Fis Protein in Replication of the Escherichia coli Chromosome MARCIN FILUTOWICZ,* WILMA ROSS, JADWIGA WILD, AND RICHARD L. GOURSE Department of Bacteriology, E. B. Fred Hall, 1550 Linden Drive, University of
Wisconsin-Madison, Madison, Wisconsin 53706 Received 20 August 1991/Accepted 18 November 1991
We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in thesefis mutant strains following upshift to 44°C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32°C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.
The initiation of DNA replication in Escherichia coli is a complex process which must be carried out with high accuracy under the diverse environmental conditions encountered by a cell. The initiation of DNA replication is very sensitive to modest changes in DNA supercoiling in vitro (1). Changes in DNA structure in vivo are known to occur in response to variation in temperature (20, 70), osmolarity (23), nutrients (2), or availability of oxygen (9). Therefore, the cell must have a means of compensating for the effects of alterations in DNA structure in order to carry out initiation of replication under a range of conditions. A class of abundant low-molecular-mass DNA binding proteins plays an accessory role in a variety of complex processes in vivo, including DNA replication, recombination, and transcription (see references 10, 17, and 59 for reviews). A common feature of HU and integration host factor (IHF), two extensively studied examples of these proteins, is the ability to bend the DNA to which they bind (27, 50). IHF can bind and bend oriC DNA at a unique site (15, 49). In a fractionated in vitro replitation system, both HU and IHF either stimulate replication or inhibit it in a concentration-dependent fashion (1, 5, 7, 45). E. coli produces several other small and abundant DNA binding proteins in addition to HU and IHF (10, 17, 59). In this study, we investigated the possible role of one of these, the Fis protein, in DNA replication. Fis is a 12-kDa protein which binds to DNA in a sequence-specific manner (6, 21, 28, 31, 32, 34-36, 54, 66). Fis was discovered initially as a host factor required to carry out the phage Mu gin and Salmonella typhimurium hin site-specific DNA inversion reactions in vitro (31, 34). Fis also participates in a related recombination system, cin, of phage P1 (21) and stimulates the excisive recombination of phage X (66). Fis was recently shown to activate rRNA transcription both in vivo and in vitro via interactions with an upstream activator region (54). We present in this report several lines of evidence indicating that the Fis protein plays a role in initiation of replication of the E. coli chromosome.
MATERIALS AND METHODS Bacterial strains and plasmids. E. coli strains used in this
study included the following: CSH50 [ara A(pro-lac) rpsL thi] and CSH50 fis::kan (36); MC1000 [AlacX74 araD139 A(ara-leu)7697 galU galK strA]; RJ1617 (MC1000 fis: :kan767) from Reid Johnson; CAG4000 (MG1655 AlacX74) from C. Gross and D.-J. Jin; RLG1347 (CAG4000 fis::kan) (54); EC6 (argE thi ilv) and EC8 [argE thi dnaA46(Ts)] (12); EC1510 [argE thi gyrB4J(Ts)] and EC1512 [argE thi gyrB402(Ts)] (14); and SD108 (trpE pyrF gyrB*) (51). fis::kan derivatives of some of the above strains were constructed by P1 transduction with P1 vir grown on either CSH50 fis::kan or RJ1617, with selection for kanamycin resistance (50 ,ug/ml). Throughout this work, the fis allele from CSHSOfis: :kan will be referred to asfis: :kan, and thefis allele from RJ1617 will be referred to as fis767. The newly constructed fis mutant strains include the following: ECF5201 (EC6 fis::kan), ECF5202 (EC6 fis767), ECF5203 (EC1510fis767), ECF5204 (EC1512fis767), ECF5206 (SD108 fis767), and RLG1863 (CAG4000fis767). Plasmid pLAR7 contains both the pBR322 origin of replication and oriC (53), and the isogenic plasmid pKO6 contains only the pBR322 origin (53). Plasmid pOC15 contains oriC as its only origin of replication (68). Media and growth conditions. Bacteria were grown on Luria-Bertani (LB) agar without or, where indicated, with 1% glucose. M9 complete medium used in experiments measuring DNA synthesis was supplemented with 0.5% Casamino Acids (Difco), 2% glucose, 5 ,ug of thymidine (Sigma) per ml, 250 ,g of deoxyadenosine (Sigma) per ml, and [3H]thymidine (1.8 uCi/,Lg/ml; Amersham). Transformation efficiency and plasmid segregation. Competent cells and plasmid DNAs were prepared according to published procedures (40). Plasmid segregation tests were performed as described in footnote a to Table 2. Individual colonies were examined for plasmid retention by touching a colony edge with a toothpick and transferring the material to the selective media. Fluorescence microscopy. The fluo-phase combined method of Hiraga (24) was modified as follows. Cells from a plate were suspended in a drop of 0.9% NaCl on a glass slide. The sample was heat fixed, methanol washed for 5 min, washed with tap water for 5 s, and air dried for approxi-
* Corresponding author. 398
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mately 20 min. Slides were stained with 10 RI of a 2.5-,ug/ml solution of 4',6-diamidino-2-phenylindole (Sigma) in 0.9% NaCl. A Zeiss photomicroscope with a NeoFluar 100/1, 30
Oel objective was used. Kodak Ektachrome 400 film was used for photography. DNase "footprinting." Six-microgram aliquots of pLAR7 DNA, purified on a Qiagen column (Qiagen Inc.), were digested with either SalI or HindIII, phenol extracted and ethanol precipitated, and then labeled for 5 min at 30°C with Sequenase (U.S. Biochemicals) and either [a-32P]TTP in the presence of 0.75 mM dideoxy-CTP for the SalI digest or [a-32P]dATP and 0.75 mM dideoxy-GTP for the HindIII digest. Labeled DNA was recut with either XhoI or Sail, and the fragment containing oriC was gel purified. Binding reaction mixtures (25 ,l) contained approximately 4 ng of purified DNA fragment in a solution containing 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 150 mM NaCl (or 80 mM NaCl), 1 mM dithiothreitol, 100 ,ug of bovine serum albumin per ml, and the indicated concentrations of purified Fis or IHF. Freshly prepared dilutions of Fis or IHF were made in 25 mM Tris-HCI (pH 7.5)-10% glycerol-50 mM NaCl. DNase I (Worthington) was added to a final concentration of 1 to 4 ,ug/ml and incubated for 30 s at room temperature, and the reaction was terminated by rapid phenol extraction in the presence of 10 mM EDTA. Sonicated calf thymus DNA (2.5 Vug) was added as a carrier for ethanol precipitation. Conditions of gel electrophoresis and autoradiography were as described previously (54). RESULTS fis mutants form filamentous cells at 44°C with reduced numbers of nucleoids and aberrant nucleoid segregation. E. coli strains carrying fis mutations in which a kanamycin resistance cassette was inserted into the intact fis gene (fis::kan [36]) or into a partially deletedfis gene (fis767 [32]) grow well under standard laboratory conditions (32, 36). However, we found that these fis null mutants failed to form individual colonies at 44°C on LB agar plates or at 42°C on minimal glucose plates containing Casamino Acids although there was residual growth in areas of heavy inoculum. At permissive temperatures, fis mutant strains form colonies with distinctive morphology (wrinkled edges). This feature is characteristic of E. coli mutants with perturbed cell division
(8).
Cell shape and nucleoid distribution in fis mutant and wild-type cells were compared by using phase-fluorescence microscopy (Fig. 1). Wild-type cells are short and nucleated irrespective of the temperature of growth. In contrast,fis767 andfis::kan strains display pronounced filamentation at 44°C and a lesser, although still considerable, degree of filamentation at 37 or 32°C (Fig. 1 and data not shown). The localization of DNA as judged by 4',6-diamidino-2-phenylindole staining differs in fis mutant cells grown at different temperatures. Staining in cells incubated at 44°C is very intense and distributed unevenly in the filament. Some anucleate cells are observed at 44°C. In cells grown at 37°C, DNA staining is less intense and is distributed throughout the filament. The morphological and DNA staining characteristics of the fis mutant cells at high temperatures are reminiscent of strains deficient in DNA replication (3, 8, 67). Therefore, we measured DNA synthesis directly in fis mutants at different temperatures.
DNA synthesis is inhibited in fis mutants exposed to high temperatures. The kinetics of incorporation of [3H]thymidine in wild-type cells (Fig. 2A) was compared with that in fis
399
mutant cells (Fig. 2B) at 32°C and following a shift to 44°C (time = 0). Incorporation is comparable in wild-type and mutant cells at 32°C. However, following a shift to 44°C, DNA synthesis in the fis mutant cells stops after approximately four chromosome equivalents of DNA are made (Fig. 2B). Conditional missense mutants affecting different components of the DNA replication machinery synthesize various amounts of DNA prior to inhibition. Mutants affecting DNA elongation show an immediate arrest in synthesis upon shift to nonpermissive conditions, while mutants affecting the initiation stage synthesize on average a third of a chromosome equivalent before inhibition occurs (reference 42 and references therein). The long delay in inhibition observed in fis null mutants after shift to 44°C does not resemble either of these two patterns but does resemble the inhibition observed with conditional mutants in the ,B subunit of DNA gyrase or when inhibitors of the P subunit of gyrase are used (12-14, 47, 56, 62). However, it should be noted that thefis mutation used contains a deletion of much of the fis gene, and, therefore, it does not make a temperature-sensitive protein as do the gyrase mutants. Rather, the requirement of the cell for Fis is dependent upon temperature. Rifampin inhibits resumed DNA synthesis in fis mutants following temperature downshift, but chloramphenicol does not. After prolonged incubation at 44°C, followed by downshift to 32°C, DNA synthesis resumes in fis mutant cells without a lag and appears comparable to that in wild-type cells (Fig. 2A and B). Initiation of DNA replication in vivo requires a transcriptional step (37) and de novo protein synthesis (39). We asked, therefore, whether new RNA synthesis or new protein synthesis was required in order for DNA synthesis to resume upon downshift to 32°C. As expected, in wild-type cells, which contain initiation-proficient complexes at 44°C, DNA synthesis continues, presumably at already established replication forks, when further RNA or protein synthesis is blocked by rifampin or chloramphenicol prior to downshift (Fig. 2A). Synthesis of approximately one-third of a chromosome equivalent occurs in wild-type cells before rifampin or chloramphenicol inhibits new initiations. Infis mutant cells, chloramphenicol addition 5 min prior to temperature downshift does not block renewed DNA synthesis, and approximately one chromosome equivalent of DNA is synthesized before replication rounds terminate. Thus, no new protein synthesis is required for DNA synthesis to resume, and the synthesis of approximately one chromosome equivalent of DNA (rather than one-third as observed for the wild type) suggests that the chromosomes of fis mutant cells are synchronized by prolonged exposure to 44°C. In contrast, no resumption of DNA synthesis occurs if fis mutant cells are treated with rifampin before temperature downshift. We interpret these results to mean that the temperature shift is changing the requirements for replication and that Fis is required at or before the transcriptional step in initiation of replication at 44°C. Enhanced temperature sensitivity of DNA replication in gyrB fis double mutants. fis mutants and gyrB(Ts) mutants behave similarly in the DNA synthesis experiments described above (14, 47), suggesting that the loss of Fis and inactivation of gyrase might affect the same step(s) in initiation of replication. We utilized a genetic approach to ask whether DNA synthesis is more vulnerable to elevated temperature in double mutants carrying gyrB and fis::kan alleles. Isogenic double mutant strains were constructed by P1 transduction by using either of two fis::kan alleles and
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Wild-type
fis767
320C
370C
440C
FIG. 1. Phase-fluorescence micrographs of a wild-type strain (CAG4000) and the isogenicfis767 mutant (RLG1863) grown at the indicated temperatures on LB agar supplemented with 1% glucose. Although fis mutants do not form colonies at 440C, they exhibit residual growth in areas of heavy inoculation. Wild-type or fis mutant cells from areas of heavy inoculum were prepared for microscopy by using 4',6-diamidino-2-phenylindole staining as described in Materials and Methods. Nucleoids fluoresce brightly while cells scatter light under these conditions. All panels are at the same magnification (X2,000).
Fis PROTEIN IN E. COLI CHROMOSOME REPLICATION
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B
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FIG. 2. The effect of temperature on DNA synthesis in a wild-type strain (EC6) (A) and the isogenicfis767 mutant strain (ECF5202) (B). Strains were grown at 32°C in complete M9 medium containing [3H]thymidine (as indicated in Materials and Methods). At a cell density of 107 per ml (time = 0), the culture was divided into two parts: one part was incubated further at 32°C (0), and the other was shifted to 44°C (@). At the time indicated (arrow), the cultures grown at 44°C were diluted fourfold in fresh medium prewarmed to 44°C, and the following antibiotics were added 5 min prior to the temperature downshift: none (A), 200 ,g of rifampin per ml (l), and 150 ,ug of chloramphenicol per ml (G). One-milliliter aliquots of the cultures were taken at the times indicated for determination of trichloroacetic acid-precipitable radioactivity. The optical density at 650 nm of cell cultures was monitored up to the point of temperature downshift, and no inhibition of cell growth was found for either wild-type orfis767 mutant cells (data not shown).
either of two temperature-sensitive gyrB alleles (gyrB4J or gyrB402) or the gyrB* allele. gyrB41 and gyrB402 are known to affect chromosomal supercoiling in a temperature-dependent fashion (64) and to inhibit initiation of DNA replication at nonpermissive temperatures (13, 14, 47). In addition, gyrB402 is believed to affect the elongation phase of DNA synthesis at 42°C (14). In the gyrB* mutant, overall supercoiling of the DNA is reduced by approximately 30% compared with that in wild-type cells (51). The isogenic sets of single and double mutant strains were tested for growth at different temperatures on LB plates (Fig. 3). Each of the double mutant strains shows a pattern of temperature sensitivity different from that of its fis mutant parent or its gyr mutant parent. The double mutant gyrB402 fis767 does not grow at 37°C, and the double mutant gyrB41 fis767 grows more slowly at 37°C, although temperatures above 42 or 44°C are required to prevent growth of the single mutant parent strains. The gyrB* mutation, which is not a temperature-sensitive gyrB allele, enhances the sensitivity of the fis767 mutant to elevated temperatures; the gyrB* fis767 double mutant grows poorly at 32 and 37°C and does not grow at 41°C. To determine whether the enhanced sensitivity to temper-
320C
ature in the gyrB fis double mutants is caused by the inhibition of DNA replication, the incorporation of [3H]thymidine into DNA was measured at 32°C and at 38°C in the single mutant strains (either gyrB41 or fis767) and in the double mutant gyrB41 fis767. In each of the single mutant strains, incorporation of [3H]thymidine increases exponentially with time at both temperatures (Fig. 4A and B). However, DNA synthesis is inhibited at 38°C in the gyrB41 fis767 double mutant (Fig. 4C), even though the optical density increases at this temperature (data not shown). Similar inhibition of DNA synthesis was observed with the gyrB402 fis767 double mutant strain, and the temperature required to inhibit synthesis was even lower (36°C). These results indicate that the gyrB(Ts) and the fis mutations synergistically affect DNA synthesis. DnaA protein is required for initiation of replication at oriC (26). Despite repeated attempts, we were unable to introduce the fis alleles into the EC8 [dnaA46(Ts)] strain by P1 transduction. It is important to point out, however, that this apparent incompatibility may be influenced by genetic background since a viable dnaA46fis767 double mutant has been constructed by another laboratory. However, the dou-
370 C
410 C
FIG. 3. The effect of temperature on growth of wild-type (WT) andfis and gyrB single mutant and double mutant strains. Bacteria from a single colony of the strains indicated were streaked onto LB agar plates and incubated at the indicated temperatures for 24 h.
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FIG. 4. The effect of temperature on DNA synthesis in isogenic strains gyrB41(Ts) (EC1510) (A), fis767 (ECF5202) (B), and gyrB41 fis767 (ECF5203) (C). The strains were grown at 30°C in complete M9 medium containing [3H]thymidine (as indicated in Materials and Methods), and at a cell density of 107 cells per ml, the culture was divided into two parts; one part was incubated further at 30°C (A), and the other one was shifted to 38°C (A). One-milliliter aliquots were taken at the times indicated for determination of trichloroacetic acid-precipitable radioactivity.
from the fis mutant strains is more frequent than from the isogenicfis' strains. At 30°C, pOC15 is lost two- to threefold more frequently from fis mutant than from fis+ strains. This loss is greater at high temperatures, with no detectable retention of pOC15 at 42°C in experiment A and five- to sixfold-reduced retention, compared with fis+ cells, at 42°C in experiment B. Thus, we conclude that in the absence of Fis protein, maintenance of oriC minichromosomes is further impaired, perhaps because of the direct involvement of Fis in replication. Fis protein binds to the minimal oriC. Since fis mutations affect initiation of replication, we tested whether Fis binds to the minimal oriC region in DNase I footprinting experiments. Such binding might influence oriC function by altering local DNA structure and/or by affecting interactions between oriC and other proteins. Purified Fis was found to protect two sites of relatively high affinity: Fis I, centered at position 202 and partially overlapping DnaA site R2, and Fis II, centered at approximately position 282, adjacent to DnaA site R4 (Fig. 5A to C and Fig. 6). In each of these sites, approximately 25 bp of sequence is protected. The protected regions are interrupted by two areas of enhanced cleavage (or lack of protection), separated by approximately 12 bp and offset on the two strands by 2 to 3 bp (Fig. 5A to C and Fig. 6). Positions of enhanced DNase I cleavage within Fis footprints have been described for other systems (6, 54) and are located in characteristic positions with respect to the degenerate Fis consensus sequence G/T--YR--A/T--YR--C/A (28). On the basis of the boundaries of protection at relatively low Fis concentrations and the location of enhanced or unprotected positions within Fis site I (Fig. 5A to C), we
ble mutant strain is more temperature sensitive than either the fis767 or the dnaA46 parent (30a). oriC minichromosomes are not stably maintained in fis mutants. The replication of oriC requires almost the same set of factors whether it is a part of the intact chromosome or present on an extrachromosomal plasmid (see reference 69 for a review). We asked, therefore, whether replication of oriC minichromosomes requires Fis protein. Plasmids carrying the pBR322 origin (pLAR7 and pKO6) transform each of the isogenic fis+, fis::kan, and fis767 strains used with a comparable frequency irrespective of temperature (Table 1). The oriC-dependent plasmid pOC15, however, transforms the fis mutant strains with significantly reduced efficiencies at 42°C compared with those at 30°C, while it transforms the wild-type (fis') strains with comparable efficiencies at the two temperatures. (Relatively low transformation efficiency of wild-type strains is a characteristic feature of oriC plasmids [68, 69].) The failure to transform fis mutant strains with pOC15 at 42°C is not caused by growth inhibition of recipient bacteria by extra copies of oriC and/or overreplication of oriC, since the plasmid containing both oriC and the pBR322 origin can be efficiently transformed under these conditions. The stability of afis mutant pOC15 transformant obtained at 30°C was then evaluated at 42°C by using two different plasmid segregation tests (Table 2). The control plasmids pLAR7 and pKO6 are stably maintained irrespective of host and temperature of incubation. The oriC plasmid pOC15, as previously shown (references 15 and 69 and references therein) segregates frequently in wild-type cells irrespective of temperature. However, the loss of the pOC15 plasmid
TABLE 1. Reduced transformation frequency of fis mutants with oriC plasmid pOC15 Recipient strain at tempa
fis+
Plasmid pKO6 pLAR7 pOC15
fis767
fis::kan
300C
42°C
300C
42°C
1.5 x 10-4 2.1 x 10-4 1.2 x 10-6
1.6 x 10-4 1.9 X 10-4 1.3 x 10-6
1.2 x 10-4 1.9 X 10-4 1.7 x 10-6
1.7 x 10-4 2.4 x 10-4
