Abstract
Studies of two temperature-sensitive Escherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity. We have therefore reexamined the molecular basis of the temperature sensitivity of strain AS17. We find that the supD allele in this strain had lost its temperature sensitivity. The temperature sensitivity of the strain, in media of all osmolarity, results from the synthesis of a mutant DNA topoisomerase I that is itself temperature-sensitive. Nucleotide sequencing of the AS17 topA allele and studies of its expected cellular product show that the mutant enzyme is not as active as its wild-type parent even at 30 degrees C, a permissive temperature for the strain, and its activity relative to the wild-type enzyme is further reduced at 42 degrees C, a nonpermissive temperature. Our results thus implicate an indispensable role of DNA topoisomerase I in E. coli cells grown in media of any osmolarity.
Highlights
E. coli DNA topoisomerase I relaxes negatively supercoiled DNA [4] and has a key role in the modulation of intracellular DNA supercoiling [1,2,3]
Studies of two temperature-sensitive Escherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity
Temperature-dependent expression of E. coli topA was accomplished in strains AS17 and BR83, which were constructed by the introduction of an amber mutation in topA and the expression of a plasmid-borne or chromosomally located temperature-sensitive1 amber-suppressor
Summary
Identification of Mutations in the topA Gene of Strain AS17—The topA region of DNA from strain AS17 (FϪ topA17(am) pLL1(Tet® supD43,74)) cells was amplified by the polymerase chain reaction (PCR), using a pair of primers 5Ј-AAT-CCG-CTC-GAG-CTC-GTT-GCCAGT-GGA-AGG-TTT-3Ј and 5Ј-GGC-TAG-TCT-AGA-CCA-CTA-TATCAT-TTA-TAG-CCT-3Ј. Rapid Lysis of Cells and Isolation of Plasmids for Analyses of Linking Number Distributions—E. coli DM800 ⌬topA cells were sequentially transformed with pBR322 and a pBeloBAC11 derivative expressing wild-type or the G65N/W79S or Y319A mutant DNA topoisomerase I from a lac promoter. When the cultures reached an apparent optical density of 0.4 – 0.6 at 595 nm, 5-ml portions of each were placed in two sets of 50-ml tubes, one kept in the 30 °C shaker and the other placed in a 42 °C shaker water-bath After another 10 min, rapid lysis of cells was performed by pouring into each culture an equal volume of preheated lysis buffer (80 °C) containing 3% SDS and 0.2 M NaOH [14]. A 32P-labeled DNA probe, prepared by random priming of an EcoRI-EagI fragment from the tet region of pBR322, was used to selectively detect pBR322 by Southern hybridization
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