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Journal of Bacteriology, February 2002, p. 1214-1218, Vol. 184, No. 4
0021-9193/01/$04.00+0     DOI: 10.1128/jb.184.4.1214-1218.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction and Synchronization of dnaA Temperature-Sensitive Mutants of Streptomyces

Li-Fong Lee, Shun-Hua Yeh, and Carton W. Chen*

Institute of Genetics, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan

Received 23 August 2001/ Accepted 20 November 2001


   ABSTRACT
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Temperature-sensitive mutants of Streptomyces defective in initiation of chromosome replication were created by in vitro site-directed mutagenesis in the dnaA gene followed by gene replacement. When they were shifted to 39°C replication in the mutants ceased in about 90 min but resumed on return to 30°C. This allowed manipulations to achieve replication synchronization.


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The filamentous Streptomyces bacteria possess linear chromosomes (13). These linear DNA molecules contain long terminal inverted repeats and covalently bound terminal proteins at the 5" ends. Replication is initiated at the internal oriC at the center of the chromosomes and proceeds bidirectionally towards the termini (15). The oriC sequence has been identified in several Streptomyces species (3, 11, 17) and is located between the dnaA and dnaN genes in the conserved rnpA-rpmH-dnaA-dnaN-recF-gyrB gene cluster, like in many other eubacteria.

DnaA controls the initiation of replication by forming an open initiation complex through extensive interactions with the DnaA boxes in oriC. The DnaA proteins of Streptomyces, like those of other bacteria, contain the following four domains: a relatively divergent N-terminal sequence, a flexible linker, a conserved ATP-binding domain, and a DNA-binding region at the C terminus (reviewed in reference 16). The DnaA protein of Streptomyces lividans (73 kDa) contains an extra stretch of about 120 (predominantly acidic) amino acids in the second domain, the significance of which is not clear. Transcription of Streptomyces dnaA is auto-regulated by the DnaA protein in vivo (14).

Temperature-sensitive mutations in dnaA [dnaA(Ts)] that are defective in initiation of chromosome replication have been isolated from several bacteria. Most dnaA(Ts) mutants also exhibit derepression of dnaA gene expression, and many are suppressed by mutations in the RNA polymerase ß subunit in an allele-specific manner (1). The latter suggests the presence of important interactions between DnaA proteins and RNA polymerase during replication initiation.

The dnaA(Ts) mutants are useful not only for studies of chromosome replication but also for synchronization of the culture. So far, no dnaA(Ts) mutants have been available for Streptomyces. Flett et al. (7) isolated four mutants of Streptomyces coelicolor defective in chromosome replication at 39°C. Only one of the mutants was defective in initiation of chromosome replication, but the defect was in dnaE, which encodes a polIII DNA polymerase subunit (6).

Instead of screening for dnaA(Ts) mutants in the classical fashion, we decided to construct them by site-directed mutagenesis based on the known mutations of this kind in Escherichia coli (9), most of which are within the conserved C terminus of DnaA (Fig. 1). Of these amino acid substitutions, the following three were chosen to be imitated in Streptomyces: (i) the A184V mutation in the ATP binding motif found (accompanied by another substitution) in six mutants, (ii) the H252Y mutation present (in combination with other substitutions) in a Ts and a cold-sensitive mutant, and (iii) the M441T mutation in the DNA binding region present in one mutant. The corresponding substitutions in the DnaA protein of S. lividans would be A369V, H437Y, and M599T, which are used in this paper.



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  		FIG. 1. Temperature-sensitive mutations in the conserved regions of DnaA. The genetic organization around oriC (open box) of the S. lividans chromosome is shown above. The arrows indicate the location and direction of transcription of the relevant genes. The conserved C terminus of the Streptomyces DnaA protein aligned with that of the E. coli homolog is shown in the enlarged view. Identical amino acid residues are shaded in black. The ATP-binding domain and the DNA-binding region are indicated by lines over the sequences. The amino acid residues above the E. coli DnaA sequence depict amino acid substitutions identified in the dnaA(Ts) mutants (9). The three residues chosen for mutagenesis in Streptomyces in this study are boxed.

 
A 3.1-kb EcoRI-SphI DNA fragment containing dnaA of S. lividans (from plasmid pFF915, a gift from F. Flett and C. Smith) was ligated to EcoRI- and SphI-digested pALTER-1 vector (Promega), and site-directed mutagenesis of the dnaA gene was performed using the Altered Sites II system (Promega). The design of the three mutagenesis primers (Table 1) was such that either the nucleotide substitutions would create a new restriction site or an additional nucleotide substitution was introduced nearby to generate a new site without altering the encoded amino acids. These new restriction sites would facilitate subsequent screening for the mutants. For the A369V mutation, a CC dinucleotide at nucleotides (nt) 1,106 and 1,107 (from the initiation codon) was converted to TG, resulting in a new PmlI/BsaAI restriction site (Fig. 2A). For the H437Y mutation, a C-to-T substitution at nt 1,309 was created. In addition, a C-to-A substitution was introduced at nt 1,302 to generate a new EcoRI site. For the M599T mutation, a T-to-C transition at nt 1,796 was accompanied by a C-to-T substitution at the preceding codon, which generated a new SnaBI/BsaAI site without altering the Ala codon.


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  		TABLE 1. Primers used for site-directed mutagenesis

 


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  		FIG. 2. Replacement of the wild-type dnaA with the mutation alleles. (A) The three mutant alleles created in vitro. The relevant stretches of the wild-type (WT) and mutant nucleotide sequences (numbered above) and the encoded amino acids are aligned. The nucleotide substitutions are shaded in black, and the amino acid substitutions are shown in italics. The new restriction sites created by the substitutions in the mutant sequences are underlined. (B) The 5.0-kb ScaI-StuI fragment (open box) containing the M599T mutation (at the SnaBI site) was placed on the pMTL23 vector (4) along with tsr (open arrow) and the melC operon (filled arrow). The resulting plasmid (pDANSTm) was used to transform S. lividans TK64 and 1326 to produce mutants that have undergone double crossovers (dashed crosses) between the homologous dnaA sequences on the vector and the chromosome. The thin arrow in the ScaI-StuI fragment indicates the dnaA coding sequence. The 3.5-kb hybridization probe used for mutation confirmation is indicated by the thick bar above and the expected hybridization fragments marked by the brackets and their sizes (in kilobases). (C) Confirmation of mutation in the transformants. Genomic DNA of LL1 (a representative) was isolated, digested with BamHI and SnaBI, separated by electrophoresis, and hybridized with the DNA probe (see panel B). Many transformants exhibited the same hybridization pattern as TK64 (not shown).

 
In order not to disturb gene expression in the dnaA-oriC region, gene replacement was performed without inserting any selection marker in the final products. The 3.1-kb EcoRI-SphI fragment containing each mutant allele was used to restore a 5.0-kb ScaI-StuI fragment (spanning from rnpA to dnaN), which was placed on an E. coli vector next to the thiostrepton (Thio) resistance gene (tsr) and the melanin operon (melC) (Fig. 2B). The suicide plasmids were used to transform S. lividans TK64 (10). Thior and Mel+ transformants (containing integration of the suicide vector via a single crossover) were isolated and then allowed to sporulate in the absence of Thio. The spores were grown on solid medium supplemented with copper and tyrosine, and melanin-lacking colonies (4 to 20%) were isolated and found to be Thios, as was expected for the loss of the vector DNA through a second crossover. The double crossover would have either restored the wild-type sequence or replaced the wild-type sequence with the mutant allele.

For the M599T mutation, 12 Ts cultures (failing to grow at 39°C) were found among 32 Mel- Thios cultures tested. When digested with BamHI and SnaBI and probed with dnaA DNA, the Ts mutants exhibited a 2.5- and a 1.0-kb fragment as expected for the presence of the mutant allele (Fig. 2C). The remaining 20 cultures showed a 3.5-kb hybridization fragment as did the parent TK64. All 12 mutants grew and sporulated normally at 30°C but failed to grow at 39°C on four different solid media, Luria broth, R5 (12), CM (5), and CMS (CM supplemented with 0.3 M sucrose). One mutant, designated LL1, was chosen for further characterization. The mutant allele (M599T plus the new SnaBI site) in LL1 was designated dnaA211, following the allele designation in E. coli. The same procedure allowed the creation of dnaA211 temperature-sensitive mutants from wild-type S. lividans 1326 (LL2) and S. coelicolor M145 (LL4).

For the other two mutant alleles, no temperature-sensitive mutants could be created. All the Mel- Thios recombinants (45 for A369V and 87 for H437Y) screened had restored the wild-type genotype (data not shown), suggesting that these mutations might be lethal. In the dnaA(Ts) mutants of E. coli, every A184V and H252Y substitution was accompanied by at least one extra substitution elsewhere in DnaA (9). Perhaps these mutations in E. coli and their counterparts (A363V and H437Y) in Streptomyces are lethal without an intragenic suppressor.

During the construction of the M599T dnaA(Ts) mutants, the intermediate Thior Mel+ transformants (containing integrated plasmid) exhibited only slight temperature sensitivity (poor growth at 39°C). Many E. coli dnaA(Ts) mutations (including dnaA211) are defective in auto-repression of dnaA (summarized in reference 9). It was possible that the DnaA hetero-oligomers in Streptomyces failed to repress dnaA and that the overexpression of the DnaA protein suppressed the decreased activity of the DnaA proteins. Suppression of dnaA(Ts) mutations by polyploidy has been observed in E. coli (9).

Incorporation of [3H-methyl]thymidine into alkaline-resistant and acid-precipitated material was used to monitor chromosome replication in LL1 in liquid medium (Fig. 3). At 30°C replication continued into the stationary phase at a rate slightly lower than that of TK64. Upon shift to 39°C, DNA synthesis continued for about 2 h and then ceased. Optical density, however, continued to increase, even after the chromosome replication had stopped (Fig. 3). This showed the independence of protein synthesis on replication in Streptomyces as in E. coli. The parent TK64, in contrast, showed no arrest of replication at 39°C. The rate of replication and growth for this culture actually increased slightly at 39°C.



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  		FIG. 3. Arrest of replication in LL1 at 39°C. Spores of LL1 (left panel) and TK64 (right panel) were inoculated in YPGS medium (7) with added [3H-methyl]thymidine at 30°C to an initial optical density at 600 nm (OD600) of 0.015. At early log phase (arrows), the cultures were shifted to 39°C, and incubation was continued (t = 0 h). Optical density (filled circles) and incorporation of [3H-methyl]thymidine into alkaline-resistant and acid-insoluble material (filled triangles) were monitored throughout the period. For comparison, the same cultures were grown at 30°C without temperature shifts (open symbols). The slightly faster growth and thymidine incorporation of TK64 at 39°C (right panel) was reproducible.

 
The arrest of replication initiation in LL1 at the nonpermissive temperature was reversible. After incubating at 39°C for as long as 6 h, growth resumed with little or no delay on shifting back to 30°C. This was used to synchronize the chromosome replication in the dnaA(Ts) mutants. Logarithmically growing LL1 cultures were shifted to 39°C for 3 h to stop replication completely and then returned to 30°C for 10 min before shifting to 39°C again (Fig. 4). Replication resumed immediately at 30°C and continued at 39°C. The final level of thymidine incorporation increased by about 85% of the level reached by the previous round of replication, indicating that 85% of the chromosomes had initiated replication within the 10-min period. The time required for the new round of replication was about 90 to 100 min.



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  		FIG. 4. Synchronization of LL1. Spores of LL1 were inoculated in YPGS medium with added [3H-methyl]thymidine at 30°C. Incorporation of [3H-methyl]thymidine into acid-insoluble material was monitored. At t = 0 h (early log phase), the culture was shifted to 39°C for 3 h before switching back to 30°C for 10 min (shaded area). At the end of 10 min, the culture was returned to 39°C (•). For comparison, one culture remained at 39°C throughout without switching to 30°C ({circ}).

 
If the initiation at the permissive temperature was allowed for 30 min, the increase of thymidine incorporation was about twofold, indicating that essentially all the chromosomes had been replicated, and the new round of replication also took about 90 to 100 min (data not shown). The longer initiation time period compromised the degree of synchrony but allowed more cells to participate in synchronization.

The quick resumption of the replication initiation upon return to 30°C after the arrest at 39°C was different from most dnaA(Ts) mutants of E. coli, which require a small lag before reinitiation (8). The dnaE mutant of S. coelicolor, defective in initiation or replication at 39°C, also exhibited a lag of about 2 h before replication was resumed at the permissible temperature (6, 7). The reason for the immediate reinitiation of chromosomal replication in LL1 is not clear, but it probably reflects the fast restoration of activity of the existing DnaA211 proteins.

The rate of thymidine incorporation gave estimates of the doubling time (generation time) of these mycelial cultures. The doubling time for TK64 was about 105 min at 30°C and 95 min at 39°C (Fig. 3). In comparison, the doubling time for LL1 was 115 min at 30°C. From the synchronization experiment (Fig. 4), the time for the replication of the chromosome was estimated (from initiation of replication in the 10-min time frame at 30°C to completion at 39°C) to be 90 to 100 min. Taking into account the size of the chromosome (8 Mb) and the number of replication forks (two), this would give a chain growth rate of polymerization of about 670 to 740 nt/s at 39°C. This is comparable to the DNA chair growth rate of 560 nt/s for E. coli growing at a doubling time of 100 min at 37°C (2).


   ACKNOWLEDGMENTS
 
This research was supported by a research grant (NSC89-2312-B010-002) to C.W.C. from R.O.C. National Science Council. C.W.C. is a recipient of a research award from the Medical Research and Advancement Foundation in memory of Chi-Shuen Tsou.

We thank Fiona Flett and Colin Smith (UMIST, Manchester, United Kingdom) for plasmids pFF915 and pDJ3 containing the dnaA DNA of S. lividans and Hans Bremer for critical reading of the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Genetics, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan. Phone: 886-2-2826-7040. Fax: 886-2-2826-4930. E-mail: cwchen{at}ym.edu.tw. Back


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Journal of Bacteriology, February 2002, p. 1214-1218, Vol. 184, No. 4
0021-9193/01/$04.00+0     DOI: 10.1128/jb.184.4.1214-1218.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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