Viability of Escherichia coli topA Mutants Lacking DNA Topoisomerase I
2004; Elsevier BV; Volume: 280; Issue: 1 Linguagem: Inglês
10.1074/jbc.m411924200
ISSN1083-351X
AutoresVera A. Stupina, James C. Wang,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoThe viability of the topA mutants lacking DNA topoisomerase I was thought to depend on the presence of compensatory mutations in Escherichia coli but not Salmonella typhimurium or Shigella flexneri. This apparent discrepancy in topA requirements in different bacteria prompted us to reexamine the topA requirements in E. coli. We find that E. coli strains bearing topA mutations, introduced into the strains by DNA-mediated gene replacement, are viable at 37 or 42 °C without any compensatory mutations. These topA- cells exhibit cold sensitivity in their growth, however, and this cold sensitivity phenotype appears to be caused by excessive negative supercoiling of intracellular DNA. In agreement with previous results (Zhu, Q., Pongpech, P., and DiGate, R. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9766–9771), E. coli cells lacking both type IA DNA topoisomerases I and III are found to be nonviable, indicating that the two type IA enzymes share a critical cellular function. The viability of the topA mutants lacking DNA topoisomerase I was thought to depend on the presence of compensatory mutations in Escherichia coli but not Salmonella typhimurium or Shigella flexneri. This apparent discrepancy in topA requirements in different bacteria prompted us to reexamine the topA requirements in E. coli. We find that E. coli strains bearing topA mutations, introduced into the strains by DNA-mediated gene replacement, are viable at 37 or 42 °C without any compensatory mutations. These topA- cells exhibit cold sensitivity in their growth, however, and this cold sensitivity phenotype appears to be caused by excessive negative supercoiling of intracellular DNA. In agreement with previous results (Zhu, Q., Pongpech, P., and DiGate, R. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9766–9771), E. coli cells lacking both type IA DNA topoisomerases I and III are found to be nonviable, indicating that the two type IA enzymes share a critical cellular function. The type IA subfamily of DNA topoisomerases, some of the extensively studied examples are E. coli DNA topoisomerases I and III, yeast DNA topoisomerase III, Drosophila, and mammalian DNA topoisomerases IIIα and IIIβ, are found in all living organisms (1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar, 2Wang J.C. Nat. Rev. Mol. Cell. Biol. 2002; 3: 430-440Crossref PubMed Scopus (1916) Google Scholar, 3Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2218) Google Scholar). This universal presence suggests a key cellular role of these enzymes that cannot be fulfilled by either the type IB or type II subfamily of DNA topoisomerases. In support of this notion, the viability is compromised upon inactivation of both type IA enzymes of Escherichia coli (4Zhu Q. Pongpech P. DiGate R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9766-9771Crossref PubMed Scopus (67) Google Scholar) or inactivation of the sole type IA enzyme of the fission yeast Schizosaccharomyces pombe (5Goodwin A. Wang S.W. Toda T. Norbury C. Hickson I.D. Nucleic Acids Res. 1999; 27: 4050-4058Crossref PubMed Scopus (98) Google Scholar, 6Maftahi M. Han C.S. Langston L.D. Hope J.C. Zigouras N. Freyer G.A. Nucleic Acids Res. 1999; 27: 4715-4724Crossref PubMed Scopus (69) Google Scholar). For the budding yeast Saccharomyces cerevisiae, Δtop3 cells lacking DNA topoisomerase III are viable, but they exhibit a complex phenotype including slow growth, high level of recombination, high sensitivity to DNA-damaging agents, and inability to produce viable spores (7Wallis J.W. Chrebet G. Brodsky G. Rolfe M. Rothstein R. Cell. 1989; 58: 409-419Abstract Full Text PDF PubMed Scopus (454) Google Scholar, 8Gangloff S. de Massy B. Arthur L. Rothstein R. Fabre F. EMBO J. 1999; 18: 1701-1711Crossref PubMed Scopus (107) Google Scholar, 9Nitiss J.L. 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U. S. A. 2001; 98: 5717-5721Crossref PubMed Scopus (97) Google Scholar, 14Kwan K.Y. Moens P.B. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2526-2531Crossref PubMed Scopus (62) Google Scholar). Insight into the molecular roles of the type IA enzymes came mostly from studies of microorganisms (reviewed in Refs. 1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar, 2Wang J.C. Nat. Rev. Mol. Cell. Biol. 2002; 3: 430-440Crossref PubMed Scopus (1916) Google Scholar, 3Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2218) Google Scholar and 15Tse-Dinh Y.C. Biochim. Biophys. Acta. 1998; 1400: 19-27Crossref PubMed Scopus (52) Google Scholar). For the two E. coli enzymes, there is strong biochemical and genetic evidence that DNA topoisomerase I has a major role in the preferential removal of negative supercoils in intracellular DNA, especially in regions behind the transcribing assemblies tracking along DNA (15Tse-Dinh Y.C. Biochim. Biophys. Acta. 1998; 1400: 19-27Crossref PubMed Scopus (52) Google Scholar, 16Drlica K. Mol. Microbiol. 1992; 6: 425-433Crossref PubMed Scopus (289) Google Scholar, 17Wang J.C. Lynch A.S. Curr. Opin. Genet. Dev. 1993; 3: 764-768Crossref PubMed Scopus (111) Google Scholar, 18Luttinger A. Mol. Microbiol. 1995; 15: 601-606Crossref PubMed Scopus (74) Google Scholar, 19Zechiedrich E.L. Khodursky A.B. Bachellier S. Schneider R. Chen D. Lilley D.M. Cozzarelli N.R. J. Biol. Chem. 2000; 275: 8103-8113Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). In an in vitro plasmid replication system, DNA topoisomerase III but not I was shown to support the unlinking of the parental DNA strands to yield separate progeny DNA rings (20Hiasa H. Marians K.J. J. Biol. Chem. 1994; 269: 32655-32659Abstract Full Text PDF PubMed Google Scholar), implicating a role of DNA topoisomerase III in unlinking of DNA strands during replication (20Hiasa H. Marians K.J. J. Biol. Chem. 1994; 269: 32655-32659Abstract Full Text PDF PubMed Google Scholar). E. coli ΔtopB mutants lacking DNA topoisomerase III exhibit no growth defect (21Schofield M.A. Agbunag R. Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 5168-5170Crossref PubMed Google Scholar), however, and thus it appears that copying the parental strands might precede their complete unraveling, so that the plectonemically wound parental strands could be converted to a pair of intertwined double helices for decatenation by a type II DNA topoisomerase (22Sundin O. Varshavsky A. Cell. 1981; 25: 659-669Abstract Full Text PDF PubMed Scopus (240) Google Scholar). That the two E. coli type IA enzymes appear to play distinct cellular roles does not preclude the possibility, however, that they may also share a common function. In the yeasts, mutations in a number of genes involved in homologous recombination were shown to suppress the phenotype of yeast Δtop3 mutants, implicating a role of the topoisomerase in the resolution of a structure or structures formed in homologous recombination (23Shor E. Gangloff S. Wagner M. Weinstein J. Price G. Rothstein R. Genetics. 2002; 162: 647-662Crossref PubMed Google Scholar, 24Oakley T.J. Goodwin A. Chakraverty R.K. Hickson I.D. DNA Repair (Amst.). 2002; 1: 463-482Crossref PubMed Scopus (45) Google Scholar). The growth defect of an E. coli topA topB double mutant was also reported to be suppressed by an additional mutation in recA, a key gene in homologous recombination (4Zhu Q. Pongpech P. DiGate R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9766-9771Crossref PubMed Scopus (67) Google Scholar). Based on a comparison of the mechanisms of different subfamilies of DNA topoisomerases, it has been suggested that a candidate for such a structure might be the double Holliday junction (2Wang J.C. Nat. Rev. Mol. Cell. Biol. 2002; 3: 430-440Crossref PubMed Scopus (1916) Google Scholar, 14Kwan K.Y. Moens P.B. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2526-2531Crossref PubMed Scopus (62) Google Scholar). In contrast to the dispensability of E. coli DNA topoisomerase III in terms of cell viability (21Schofield M.A. Agbunag R. Michaels M.L. Miller J.H. J. Bacteriol. 1992; 174: 5168-5170Crossref PubMed Google Scholar), inactivation of E. coli DNA topoisomerase I was generally thought to be lethal. Shortly after the identification of a set of viable E. coli ΔtopA mutants (25Sternglanz R. DiNardo S. Voelkel K.A. Nishimura Y. Hirota Y. Becherer K. Zumstein L. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2747-2751Crossref PubMed Scopus (200) Google Scholar), it was found that the ΔtopA locus of these mutants could not be readily transduced into strain PLK831 (ΔtrpE63 pyrF287 nirA trpR72 iclR7 gal25 rpsL195) by phage P1 (26DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 27Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Abstract Full Text PDF PubMed Scopus (241) Google Scholar). The same recipient strain became more easily transduced, however, if it had acquired a compensatory change within certain regions of the E. coli chromosome, including gyrA and gyrB, which encode the two subunits of gyrase, and a region containing tolC, which encodes an outer membrane transporter (26DiNardo S. Voelkel K.A. Sternglanz R. Reynolds A.E. Wright A. Cell. 1982; 31: 43-51Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 27Pruss G.J. Manes S.H. Drlica K. Cell. 1982; 31: 35-42Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 28Trucksis M. Golub E.I. Zabel D.J. Depew R.E. J. Bacteriol. 1981; 147: 679-681Crossref PubMed Google Scholar, 29Raji A. Zabel D.J. Laufer C.S. Depew R.E. J. Bacteriol. 1985; 162: 1173-1179Crossref PubMed Google Scholar). These results suggested that ΔtopA is lethal in the absence of compensatory genetic changes. A more direct demonstration of the lethality of topA deletion was provided by experiments demonstrating the thermal sensitivity of E. coli topA amber mutants expressing a thermal sensitive amber suppressor. Presumably, suppression of the amber codon at a permissive temperature would provide an adequate level of DNA topoisomerase I, but inactivation of the suppressor at 42 °C would abolish the synthesis of a functional DNA topoisomerase I and, consequently, cell growth (30Zumstein L. Wang J.C. J. Mol. Biol. 1986; 191: 333-340Crossref PubMed Scopus (62) Google Scholar, 31Dorman C.J. Lynch A.S. Bhriain N.N. Higgins C.F. Mol. Microbiol. 1989; 3: 531-540Crossref PubMed Scopus (45) Google Scholar). The idea that the topA gene is essential for cell growth was not reinforced, however, by studies in Salmonella typhimurium, a bacterium closely related to E. coli. Early studies indicated that null mutations in an S. typhimurium gene termed supX, which was later shown to be identical to topA (28Trucksis M. Golub E.I. Zabel D.J. Depew R.E. J. Bacteriol. 1981; 147: 679-681Crossref PubMed Google Scholar, 32Margolin P. Zumstein L. Sternglanz R. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5437-5441Crossref PubMed Scopus (40) Google Scholar), led to no lethal effects; there was no indication that viability of these topA cells depended on the presence of a compensatory mutation (33Dubnau E. Lenny A.B. Margolin P. Mol. Gen. Genet. 1973; 126: 191-200Crossref PubMed Scopus (10) Google Scholar, 34Richardson S.M. Higgins C.F. Lilley D.M. EMBO J. 1984; 3: 1745-1752Crossref PubMed Scopus (82) Google Scholar). More recently, Shigella flexneri topA null mutants were also found to be viable without apparent compensatory mutations (35Bhriain N.N. Dorman C.J. Mol. Microbiol. 1993; 7: 351-358Crossref PubMed Scopus (47) Google Scholar). Thus there appears to be a difference in topA requirement in different bacteria; it is this apparent difference that prompted us to reexamine the viability of E. coli mutants lacking DNA topoisomerase I. Targeted Gene Replacement and Phage P1 Transduction—The vector pRM4-N for gene replacement in E. coli by chi-stimulated homologous recombination (40Dabert P. Smith G.R. Genetics. 1997; 145: 877-889Crossref PubMed Google Scholar) was kindly provided by Dr. G. Smith (University of Washington). In this plasmid, a multiple cloning site is placed in between two triplets of chi sequences. DNA segments derived from a 12.1-kb SalI-HindIII fragment containing the E. coli cysB-topA-trpE region (42Wang J.C. Becherer K. Nucleic Acids Res. 1983; 11: 1773-1790Crossref PubMed Scopus (37) Google Scholar) were placed within the multiple cloning site according to routine cloning manipulations (see "Results" for a description of the three classes of derivatives, all of which have a chloramphenicol resistance marker inserted at a unique SphI site of the 12.1-kb segment). The chloramphenicol resistance marker cam was amplified from pACYC184 (supplied by New England Biolabs) by PCR in the presence of primers that would add SphI sites to both ends of a 1.41-kb DNA product; the inducible lac promoter used in the construction of one class of derivatives was obtained from pJW312 as a HindIII-EcoRI fragment (30Zumstein L. Wang J.C. J. Mol. Biol. 1986; 191: 333-340Crossref PubMed Scopus (62) Google Scholar). Codon substitutions within the topA region were done by replacing appropriate DNA segments by the corresponding segments containing the alterations L5F C662H (36Zhu C.X. Qi H.Y. Tse-Dinh Y.C. J. Mol. Biol. 1995; 250: 609-616Crossref PubMed Scopus (21) Google Scholar) or G65N W79S (37Wang Y. Lynch A.S. Chen S.J. Wang J.C. J. Biol. Chem. 2002; 277: 1203-1209Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Various pRM4-N derivatives were linearized by digestion with NotI. The large linear DNA segments lacking the replication origin of pRM4-N were purified by gel electrophoresis and used for gene replacement in the cysB-topA-trpE region of the E. coli chromosome, through homologous recombination mediated by recBCD, as described by Dabert and Smith (40Dabert P. Smith G.R. Genetics. 1997; 145: 877-889Crossref PubMed Google Scholar), or the phage λ Red recombination system, as described by Datsenko and Wanner (41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar). In the latter case, the thermal sensitive plasmid expressing the λ Red functions γ, β, and exo, pKD46, was kindly provided by Dr. B. L. Wanner (Purdue University). cam+ colonies were first selected in these experiments, and several tests were utilized to ascertain the incorporation of the expected changes in chromosomal DNA samples extracted from selected colonies. For mutants in which the topA promoter was replaced by a lac promoter, or mutants in which a large topA segment was deleted, amplification of these shortened regions by PCR was carried out to confirm the presence of these altered regions and the absence of the corresponding wild-type segments. Appropriate restriction digests were also analyzed by Southern blot hybridization for the presence of the cam marker and other changes, taking advantage of alterations in restriction sites in the G65N W79S mutants (37Wang Y. Lynch A.S. Chen S.J. Wang J.C. J. Biol. Chem. 2002; 277: 1203-1209Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In all cases, the selected cam+ colonies were found to have correctly incorporated the intended changes in the topA region. Transduction by phage P1vir was carried out according to the protocol described by Silhavy (51Silhavy T.J. Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1984: 107-112Google Scholar), using a phage stock kindly provided by Dr. N. Kleckner (Harvard University). Analysis of Plasmid Linking Number Distributions—Two-dimensional agarose gel electrophoresis and the use of Southern blot hybridization to monitor plasmid supercoiling were carried out as described previously (43Stupina V.A. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8608-8613Crossref PubMed Scopus (18) Google Scholar). Construction of E. coli topA Conditional Mutants by DNA-mediated Gene Replacement—The introduction of methods for DNA-mediated gene targeting in E. coli has made it possible to construct better defined E. coli topA mutants. In order to minimize the possibility of inadvertently acquiring compensatory mutations in these mutants, conditional topA mutants were first constructed under permissive conditions that produced a functional mutant DNA topoisomerase I. Fig. 1 depicts the common features of the cloned DNA segments used for gene replacements in the topA region of the E. coli chromosome. A cam gene encoding chloramphenicol resistance was first inserted at an SphI site within a cloned 12.1-kb SalI-HindIII fragment. This insertion does not disrupt any of the open reading frames in the topA region; to minimize plausible interference with topA expression, cam transcription was also chosen to be directing away rather than into topA. Two classes of conditional topA mutants were constructed. The first class, topA ts (L5F C662H) and topA ts (G65N W79S), encodes two thermal sensitive DNA topoisomerase I with the indicated point mutations L5F C662H and G65N W79S, respectively. The L5F C662H mutant was derived from site-directed mutagenesis of individual codons of cysteinyl residues that are involved in Zn(II) binding (36Zhu C.X. Qi H.Y. Tse-Dinh Y.C. J. Mol. Biol. 1995; 250: 609-616Crossref PubMed Scopus (21) Google Scholar). The mutation C662H was found to give a thermal sensitive enzyme (36Zhu C.X. Qi H.Y. Tse-Dinh Y.C. J. Mol. Biol. 1995; 250: 609-616Crossref PubMed Scopus (21) Google Scholar), and sequencing of the mutant gene revealed the presence of a second mutation L5F in the gene, which by itself had little effect on enzyme activity. 2Y. Wang and J. C. Wang, unpublished data. The (G65N W79S) mutant was expected to produce the same mutant DNA topoisomerase I of E. coli topA strain AS17, which harbors a supD amber suppressor (30Zumstein L. Wang J.C. J. Mol. Biol. 1986; 191: 333-340Crossref PubMed Scopus (62) Google Scholar, 31Dorman C.J. Lynch A.S. Bhriain N.N. Higgins C.F. Mol. Microbiol. 1989; 3: 531-540Crossref PubMed Scopus (45) Google Scholar). The topA gene of strain AS17 has a G65N mutation and an amber codon instead of Trp-79 of wild-type topA (37Wang Y. Lynch A.S. Chen S.J. Wang J.C. J. Biol. Chem. 2002; 277: 1203-1209Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Originally, it was thought that the thermal sensitivity of the supD suppressor in this strain, which would incorporate a serine at the amber codon only at a permissive temperature (38Zengel J.M. Lindahl L. J. Bacteriol. 1981; 145: 459-465Crossref PubMed Google Scholar), was responsible for the topA ts phenotype of AS17. Recent evidence indicates, however, that the protein product in the presence of the suppressor, with amino acid changes G65N and W79S relative to the wild-type protein, is itself thermal sensitive (37Wang Y. Lynch A.S. Chen S.J. Wang J.C. J. Biol. Chem. 2002; 277: 1203-1209Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar).Table IILists of E. coli strains used in this study Listed are the strains constructed in this study.Laboratory designationaThe strains VS111, VS102, VS105, and VS106 have been deposited in the CGSCStrain backgroundtopA locusMethod of constructionVS100aAB1369topA ts (L5F C662H)bGene replacement (40, 41)VS100bMG1655topA ts (L5F C662H)bGene replacement (40, 41)VS101AB1369Plac-topA ts (L5F C662H)bGene replacement (40, 41)VS102AB1369Plac-topA ts (L5F C662H) topB::Tn5bGene replacement (40, 41),cP1vir transduction (51)VS105MG1655Plac-topA ts (L5F C662H)bGene replacement (40, 41)VS106MG1655Plac-topA ts (L5F C662H) topB::Tn5bGene replacement (40, 41),cP1vir transduction (51)VS107AB1369Plac-topA ts (G65N W79S)bGene replacement (40, 41)VS108MG1655Plac-topA ts (L5F)bGene replacement (40, 41)VS109MG1655Plac-topA ts (L5F) topB::Tn5bGene replacement (40, 41),cP1vir transduction (51)VS111aAB1369ΔtopAcP1vir transduction (51)VS111 (VS111b)MG1655ΔtopAbGene replacement (40, 41),cP1vir transduction (51)VS112AB1369top20::Tn10cP1vir transduction (51)VS113MG1655top20::Tn10cP1vir transduction (51)VS114AS17Plac-topA ts (L5F C662H)cP1vir transduction (51)VS115BR83Plac-topA ts (L5F C662H)cP1vir transduction (51)a The strains VS111, VS102, VS105, and VS106 have been deposited in the CGSCb Gene replacement (40Dabert P. Smith G.R. Genetics. 1997; 145: 877-889Crossref PubMed Google Scholar, 41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar)c P1vir transduction (51Silhavy T.J. Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1984: 107-112Google Scholar) Open table in a new tab The second class of mutant topA segments are denoted Plac-topA ts (L5F C662H) and Plac-topA ts (G65N W79S); they were derived from the corresponding members of the first class by replacing the topA regulatory region (39Tse-Dinh Y.C. Beran-Steed R.K. J. Biol. Chem. 1988; 263: 15857-15859Abstract Full Text PDF PubMed Google Scholar) with a lac promoter so that expression of the mutant topA genes can be turned on or off by the presence or absence of the lac inducer IPTG 3The abbreviation used is: IPTG, isopropyl 1-thio-β-d-galactopyranoside. (the region replaced by the lac promoter is indicated by an X in Fig. 1). An additional DNA segment bearing the mutation Plac-topA (L5F), which encodes a protein with wild-type DNA topoisomerase I activity, was also used in the construction of a control. As will be described later, several mutants with the deletion of a large region in the topA region, marked by Y in Fig. 1, were also made in the later stages of the present study. Viability of E. coli topA Mutants Expressing a Thermally Sensitive DNA Topoisomerase I—Replacement of the chromosomal topA region by topA ts (L5F C662H) or topA ts (G65N W79S), using either the chi site-dependent homologous recombination method (40Dabert P. Smith G.R. Genetics. 1997; 145: 877-889Crossref PubMed Google Scholar) or the λ Red recombination system (41Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar), was first carried out in two E. coli strains AB1369 and MG1655 (see Tables I and II for the genotypes of these and other strains used in this work). Strain AB1369 is a cysB- strain used in the original cloning of the E. coli topA gene (42Wang J.C. Becherer K. Nucleic Acids Res. 1983; 11: 1773-1790Crossref PubMed Scopus (37) Google Scholar), and strain MG1655 is a widely used laboratory strain. Transfected cell cultures were incubated at 30 °C in these experiments, except that in experiments using the λ Red recombination system the cultures were kept at 37 °C for 1 h following transfection to eliminate the thermal sensitive plasmid pKD46 bearing the λ Red genes.Table ILists of E. coli strains used in this study Listed are the strains previously reported.StrainGenotypeSource or Ref.AB1369DE(gpt-proA)62 lacY1 tsx-29 glnV44(AS) galK2(Oc) LAM- cysB38 Rac-0 hisG4(Oc) rfbD1 kdgK51 xylA5 mtl-1 argE3(Oc) thi-1A. L. TaylorMG1655F- LAM- rph-I48Guyer M.S. Reed R.R. Steitz J.A. Low K.B. Cold Spring Harbor Symp. Quant. Biol. 1981; 45: 135-140Crossref PubMed Google Scholar, 49Jensen K.F. J. Bacteriol. 1993; 175: 3401-3407Crossref PubMed Scopus (401) Google ScholarMM28galK2(Oc) LAM- IN(rrnD-rrnE)1 rpsL200(strR)M. MeselsonW3110LAM- IN(rrnD-rrnE)1 rph-1J. LedenbergBL21(DE3)hsdS gal (λc Its857 ind1 Sam7 nin5 lacUV5-T7 gene1)50Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4842) Google ScholarBL21(DE3)-topBBL21(DE3) topB::Tn520Hiasa H. Marians K.J. J. Biol. Chem. 1994; 269: 32655-32659Abstract Full Text PDF PubMed Google ScholarBR83F- topA57(Am) argA rpsL lacΔ514 supD 43, 74 (ts 42 °C)R. E. DepewAS17F- topA17(Am) pLL1[supD 43, 74 (ts 42 °C)R. E. DepewRFM480rpsL galK2 gyrB221(cour) gyrB203(ts) topA20::Tn10 Δlac7444Drolet M. Phoenix P. Menzel R. Masse E. Liu L.F. Crouch R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3526-3530Crossref PubMed Scopus (198) Google Scholar Open table in a new tab At 30 °C, the mutant enzyme with the L5H C662H or G65N W79S substitutions was expected to be fully functional; at 42 °C, the activity of the mutant enzyme would be much diminished (36Zhu C.X. Qi H.Y. Tse-Dinh Y.C. J. Mol. Biol. 1995; 250: 609-616Crossref PubMed Scopus (21) Google Scholar, 37Wang Y. Lynch A.S. Chen S.J. Wang J.C. J. Biol. Chem. 2002; 277: 1203-1209Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Surprisingly, the two topA ts derivatives of either AB1369 or MG1655 grew as well as their parent cells on agar plates at either 30 or 42 °C. To test whether DNA topoisomerase I expressed in these topA ts mutants is indeed thermal sensitive, strain AB1369 topA ts (L5F C662H) was transformed with a tetA-bearing plasmid pVS1 (43Stupina V.A. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8608-8613Crossref PubMed Scopus (18) Google Scholar), a plasmid that has been shown to be excessively negatively supercoiled ("hypernegatively supercoiled") in cells lacking DNA topoisomerase I (43Stupina V.A. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8608-8613Crossref PubMed Scopus (18) Google Scholar). Plasmid samples were recovered from a randomly selected transformant grown at 30 or 42 °C. Two-dimensional gel electrophoresis of these samples showed that the degree of negative supercoiling of the plasmid is much higher at the higher temperature (Fig. 2). Thus it appeared that these cells indeed had a much reduced level of DNA topoisomerase I at 42 °C, but despite this deficiency they grew as well as their topA+ parents. It is known, however, that DNA topoisomerase I bearing the mutations L5F C662H or G65N W79S had a significant level of residual activity at 42 °C (36Zhu C.X. Qi H.Y. Tse-Dinh Y.C. J. Mol. Biol. 1995; 250: 609-616Crossref PubMed Scopus (21) Google Scholar, 37Wang Y. Lynch A.S. Chen S.J. Wang J.C. J. Biol. Chem. 2002; 277: 1203-1209Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). To better define the requirement of DNA topoisomerase I at 42 °C, strain AB1369 and MG1655 derivatives bearing Plac-topA ts (L5F C662H) or Plac-topA ts (G65N W79S) were constructed (see Table II). In these cells, expression of DNA topoisomerase I is strongly dependent on the presence of IPTG, as demonstrated by two-dimensional gel electrophoresis of pVS1 recovered from AB1369 bearing Plac-topA ts (L5F C662H) (Fig. 3). At 30 °C, the plasmid became much more negatively supercoiled following the removal of IPTG (Fig. 3, compare lanes 1 and 2). In the presence of IPTG, the patterns at 30 and 42 °C also showed that the plasmid was more negatively supercoiled at the higher temperature, again demonstrating the thermal sensitivity of the mutant topoisomerase (Fig. 3, compare lanes 2 and 4). Plating of the mutant cells showed that even in the absence of IPTG, at 42 °C both of the Plac-topA ts derivatives grew as well as their topA+ parents, which strengthens the notion that DNA topoisomerase I is dispensable for cell growth. Cold Sensitivity of E. coli topA Mutants Lacking DNA Topoisomerase I—At 30 °C and in the absence of IPTG, the two Plac-topA ts derivatives of either strain AB1369 or MG1655 grew very poorly relative to their respective topA+ parent; comparable growth of the isogenic topA+ and topA ts strains at this lower temperature was only seen in the presence of IPTG. This strong temperature dependence of the growth of topA-deficient cells was also seen in a derivative of strain MG1655, in which the wild-type topA locus was replaced by Plac-topA(L5F). As noted earlier, DNA topoisomerase I with an L5F mutation is indistinguishable from the wild-type enzyme, at least in terms of its enzymatic activity. It was observed that MG1655 Plac-topA(L5F) grew well at either 30 or 42 °C in the presence of IPTG; in the absence of IPTG, however, a precipitous drop in topA (L5F) expression was expected, and the cells were found to grow normally at 42 °C but very poorly at 30 °C. E. coli ΔtopA Cells Are Viable at 42 or 37 °C but Not at 30 °C—In a separate series of experiments, a ΔtopA mutation, in which a large region of the topA coding sequence and the entire promoter of the gene (region indicated by Y in Fig. 1) were absent, was first introduced into strain MG1655 by DNA-mediated gene replacement at 42 °C, a temperature at which cells lacking DNA topoisomerase I are apparently viable, as described above. A transformant bearing this ΔtopA locus, as ascertained by Southern blot hybridization and PCR, was then used as the donor strain in a transduction experiment using phage P1vir. As a control, MG1655 bearing Plac-topA ts (L5F C662H), which has been shown to grow well at 42 °C in the presence or absence of IPTG, was
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