Increased Expression of the dnaA Gene Has No Effect on DNA Replication in a dnaA+ Strain of Escherichia coli

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Increased Expression of the dnaA Gene Has No Effect on DNA Replication in a dnaA

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Strain of Escherichia coli

CHURCHWARD, Gordon, HOLMANS, P., BREMER, Hans

Abstract

We have constructed a pBR322 plasmid derivative which expresses dnaA protein under the control of the E. coli lac UV5 promotor. Expression of the dnaA protein from the plasmid is inducible by isopropyl-β-D-thiogalactoside. In a dnaA⁺ strain induction has no effect on the accumulation of DNA. In contrast, in a thermosensitive dnaA46 strain, induction, at either the permissive or the nonpermissive temperature, results in an immediate stimulation of DNA accumulation. We conclude that, while in a dnaA46 strain dnaA protein limits DNA replication, in a dnaA⁺ strain dnaA protein activity does not control the timing of replication initiation.

CHURCHWARD, Gordon, HOLMANS, P., BREMER, Hans. Increased Expression of the dnaA Gene Has No Effect on DNA Replication in a dnaA

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Strain of Escherichia coli . Molecular and General Genetics , 1983, vol. 192, no. 3, p. 506-508

DOI : 10.1007/BF00392197

Available at:

http://archive-ouverte.unige.ch/unige:149825

Disclaimer: layout of this document may differ from the published version.

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Mol Gen Genet (1983) 192:50(~508

Short Communication

Increased Expression of the dnaA Gene Has No Effect on DNA Replication in a dnaA + Strain of Escherichia coil

G. Churchward 1, P. Holmans 2, and H. Bremer 2

i Department of Molecular Biology, University of Geneva, 30, Quai Ernest Ansermet, 1211 Geneva 4, Switzerland 2 University of Texas at Dallas Box 688, Richardson, Texas 75080 USA

Summary. We have constructed a pBR322 plasmid derivative which expresses dnaA protein under the control of the E. coli lac UV5 promotor. Expression of the dnaA protein from the plasmid is inducible by isopropyl-fl-D-thiogalactoside. In a dnaA + strain induction has no effect on the accumulation of DNA. In contrast, in a thermosensitive dnaA46 strain, induction, at either the permissive or the nonpermissive temperature, results in an immediate stimulation of DNA accumulation. We conclude that, while in a dnaA46 strain dnaA protein limits DNA replication, in a dnaA + strain dnaA protein activity does not control the timing of replication initiation.

Initiation of chromosome replication in E. coli requires dnaA protein both in vivo (Hirota et al. 1968) and in vitro (Fuller et al. 1982). Indirect evidence has suggested that this protein acts together with RNA polymerase in a transcriptional step in initiation (Messer et al. 1975; Bagdasar- ian et al. 1977; Filutowicz and Jonczyk 1981), prior to an event requiring the product of the dnaC gene (Zyskind et al.

1977). Strains carrying the dnaA46 allele, a temperature sensitive mutation, show reduced initiation of DNA replication, compared to dnaA + strains, at the permissive temperature, 30°C (Frey et al. 1981). Supressor mutations of the dnaA46 allele, dnaAcos, that permit growth at 42 ° C, result in increased initiation of replication at 30 ° C (Kellenberger- Gujer et al. 1978). These and other observations have led to the suggestion that the dnaA gene product controls the timing of the initiation process.

To test this idea directly, we have constructed a high copy number plasmid containing the entire dnaA gene, without its normal promotor sequences. The dnaA gene carried by the plasmid is expressed from the promotor of the E. coli lac operon, and expression can be controlled by altering the concentration of the inducer isopropyl-fl-D-thiogalactoside (IPTG) in the growth medium. In a dnaA + strain, induction of dnaA expression has no effect on DNA replication and thus we conclude that the dnaA protein does not normally limit initiation of DNA replication.

Inspection of the nucleotide sequence of the dnaA gene (E.B. Hansen etal. 1982) shows that a 1611bp HindII-- XhoI fragment contains the entire coding sequence of the dnaA protein without its promotor sequences (F.G. Hansen Offprint requests to: G. Churchward

et al. 1982). This fragment was purified after agarose gel electrophoresis of a H i n d I I - X h o I digest of pPLH302, a pBR322 plasmid carrying a PstI fragment cloned from 2tna330, a dnaA transducing phage (Hansen and von Meyenburg 1979). To construct pBC32 (Fig. 1) this dnaA fragment was ligated to the plasmid pBC30 which had been cleaved with SmaI and Sa/I. The plasmid pBC30 is a derivative of pMC1403 (Casadaban et al. 1980) into which was

cloned a 190bp HaeIII--EcoRI fragment from the plasmid pPCqb2 (Charnay et al. 1978) containing the lacUV5 promotor and the first 8 codons of the fl-galactosidase gene. The predicted size of the desired plasmid shown in Fig. 1 is 5.5 kb. Ligated DNA was introduced into HB253 by trans- formation with selection for Ap R. The HB253 strain carried an F' factor with the lael Q1 allele which results in 50-100 fold overproduction of the lac repressor protein

EcoRI

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lacZ E¢oRI

ATG D' ACC" ATG'ATT ' ACG-GAT'TCA" CTG' GGG~'AAT 'TCC"

(SmaI/HindI )

C$AA • CAA" TCA '['~1" ATG" TTT' CAG" CCT 'I'll' TCA" TTA"

~.oA

TCG" ACT" TTT'GTT • CGA" GTG. GAG' TCC' GCC, GTG

Fig. I. Structure of the dnaA overproducing plasmid pBC32. The plasmid was constructed, using standard techniques, from the DNA segments described in the text. From coordinate I to the Smal/HindII junction is lac operon DNA, from the Smal/HindII junction to the XhoI/SalI junction is the dnaA gene, and from the XhoI/SaII junction to coordinate 5545 is pBR322 DNA. The dashed arrow shows the putative Tc R protein fragment discussed in the text. The predicted nucleotide sequence around the Sinai~

HindII junction shows the position of two termination codons in phase with the lacZ initiation codon. The dnaA gene begins with a GTG codon (E.B. Hansen et al. 1982)

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507 Table 1. Escherichia coli K12 strains

Strain Relevant genotype Source

HB253 met lae (Y?) his hsr hsmK Rifa/

F'lacl Q1 lacZYA + pro + Kan R leu thyA(lac-proB) dnaA46 Cyc R leu thyA(lac-proB) dnaA + ilv A(srl-- recA) 306 srlR301 : : Tnl 0-84 LC343

LC366 JC10289

HB273 LC366 A(srl- reeA)306 srlR301 : : Tnl O-84/F'lacl ~1 lacZYA + pro + Kan R

HB275 LC366/F'lael Q1 lacZYA + pro + Kan R

HB280 LC343 A(srl--reeA) 306 srlR301 : :Tn10-84/F' lacl Q1 IaeZYA + pro + Kan R

EQ82: N. Murray W1485: L. Caro W1485: L. Caro CGSC 6195 B. Bachman P1 transduction from JC10289

P1 transduction from JCt0289

(Mfller-Hill 1975). Since we anticipated that overproduction of dnaA protein might be lethal, we tested the transformants for sensitivity to IPTG. Of 18 Ap R transformants, 7 did not grow on medium supplemented with 1 mM IPTG.

All these transformants contained plasmids of 5.5 kb, one of which was pBC32. Since the nucleotide sequence of all the segments of pBC32 have been previously reported (Sut- cliffe 1978; Charnay et al. 1978; E.B. Hansen et al. 1982;

Peden 1983) we compiled a restriction map of the plasmid based on these data. Digestion of pBC32 with EcoRI, PstI or AvaI, singly or combined, produced fragments of the predicted size (data not shown). The predicted nucleotide sequence in the region of the plac-dnaA fusion is such that translation initiating at the lacZ initiation codon is terminated before the beginning of the dnaA gene (Fig. 1).

Preliminary experiments showed that addition of I mM IPTG to cultures of HB253, carrying pBC32, had no effect on the accumulation of DNA, measured colorimetrically, but that cell growth was impaired. Colony formation on solid media was also affected by IPTG: after 24 h incuba- tion at 37 ° C, only micro-colonies were visible, compared to normal size colonies on plates without IPTG. The reason for the effect of IPTG is not clear since this inhibition was not observed in all strains. The nucleotide sequence of pBC32 predicts that the induction could result in the pro- duction of a fragment of the tetracycline resistance protein of pBR322. It seems that high level expression of either the dnaA protein or this protein fragment perturbs the growth of some strains carrying pBC32.

In order to show that the dnaA gene cloned on pBC32 is functional, the plasmid was introduced, by transforma- tion, into HB280, a dnaA46 recA- strain that carried the F" lacl Q1 plasmid, at 30 ° C. The recA deficiency was to prevent the possibility of recombination between a plasmid carrying a dnaA + allele and the chromosomal dnaA46 gene.

Colonies were resuspended and plated on ampicillin-con- raining medium at 30 ° C, and 42°C with and without IPTG. With IPTG, in all cases, the same number of colonies was observed at 42°C as at 30 ° C. No colonies were observed at 42 ° C without IPTG. These results show that upon induction with IPTG, pBC32 will direct the synthesis of a functional dnaA protein and that in the absence of IPTG, synthesis of dnaA protein from the plasmid is repressed.

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Fig. 2a-d. Effect of IPTG on DNA accumulation in strains carrying pBC32. Cultures were grown in LB medium (Lennox 1955) at 30 ° C. At an A6o 0 of 0.3 (zero time) IPTG was added to a final concentration of 1 mM to part of the culture. DNA was estimated colorimetrically (Brunschede et al. 1975). All data are normalized to 1.0 at zero time. a) HB275 dnaA + recA + b) HB273 dnaA + recA- c) HB280 dnaA46 recA- d) HB280 dnaA46 recA- : shift to 42°C at time zero. o -IPTG, • +IPTG, • +IPTG at 40 min

Since no colonies were observed at 42°C without IPTG, we can conclude that the F' factor present in the cell did not integrate and cause dnaA independent chromosomal replication (Nishimura et al. 1971) at a significant frequency under these conditions.

When pBC32 was introduced into the isogenic dnaA ÷ parent strain of HB280, either recA ÷ or recA-, no effect on DNA synthesis was observed upon the addition of IPTG (Fig. 2a, b). In HB280 (dnaA46 recA-) carrying pBC32, addition of IPTG to cells growing at 30 ° C resulted in an immediate stimulation of DNA accumulation (Fig. 2c). A shift in temperature in the absence of IPTG, from 30°C to 42 ° C, resulted in cessation of DNA accumulation; addition of IPTG at or after the shift caused DNA accumulation to continue, or resume, respectively, at an increased rate (Fig. 2d). These results show that the cloned dnaA gene is functional, that in a dnaA46 strain DNA replication is limited by the supply of active dnaA protein, but that in a wild type strain, the activity of dnaA protein does not limit DNA replication.

To explain these results, we propose that the dnaA protein acts to convert a "preinitiation complex", whose formation requires protein synthesis, to an "initiation complex". In the presence of excess dnaA protein, i.e. in dnaA ÷ strains or in the presence of IPTG in strains carrying pBC32, formation of the "preinitiation complex" deter- mines the time of initiation. In dnaA46 strains, where the activity of dnaA protein is reduced, the conversion to "initiation complex" would be delayed. This would explain the reduced DNA/mass ratio observed in dnaA46 strains (Frey et al. 1981). The observed reduction in DNA/mass ratio in dnaA46 strains compared to dnaA + strains at 30°C is approximately 30% (Frey et al. 1981). Induction of dnaA protein synthesis in a dnaA46 strain at 30 ° C would there- fore be expected to result in a comparable stimulation of replication, as was observed (Fig. 2c). In dnaAcos strains a temperature shift from 42°C to 30°C results in a burst of initiations (Kellenberger-Gujer et al. 1978). If, at 42 ° C, dnaA activity was limiting initiation, increasing the activity of dnaA protein in the cell would result in the observed increase in the rate of initiation of DNA replication.

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508

The finding of "excess capacity" for initiation in dnaA46 strains (Hansen and Rasmussen 1977) has led to the suggestion that the dnaA gene is autoregulated so that heat inactivation of the dnaA46 protein results in an induction of dnaA gene expression. The notion proposed here that a "preinitiation complex" accumulates in dnaA46 strains provides an alternative interpretation of the "excess capacity" and is consistent with the observation that heat induction of dnaA gene expression has not been observed, either at the protein level (Sakakibara and Yuasu 1982) or the mRNA level (Prentki 1983).

Identical proposals have been made previously by Ly- cett et al. (1980) to explain the stimulation of DNA synthesis observed after the addition of chloramphenicol to cultures of dnaA- bacteria. The observations described here provide evidence that these proposals are correct and that dnaA protein activity does not normally regulate DNA replication.

Acknowledgments. This work was supported by grant No 3.169.0.81 from the Swiss National Fund to L. Caro and grant GM25948 from the N.I.H. to H. Bremer. H.Bremer was supported by travelling fellowships from the Hoffmann La Roche Foundation and the Fogarty Foundation.

We should like to thank M. Visini for typing the manuscript, and O. Jenni for preparing the figures.

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Communicated by W. Arber

Received August 18, 1983

Increased Expression of the <i>dnaA</i> Gene Has No Effect on DNA Replication in a <i>dnaA</i><sup>+</sup> Strain of <i>Escherichia coli</i>

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