Effect of the deletion of the σ³²‐dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance

Abstract

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli. The P1 promoter of topA has previously been shown to be a delta 32-dependent heat-shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is > 10-fold more sensitive to heat treatment (52 degrees C) than the wild type. After brief treatment at 42 degrees C, wild-type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52 degrees C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100-fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat-survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat-inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse-labelling pattern of proteins at 42 C (displayed on SDS-polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.

Regulon and promoter analysis of theE. coliheat-shock factor, σ32, reveals a multifaceted cellular response to heat stress

“…Two newly discovered regulon members, HepA and NusB, join TopA (Qi et al 1996) as general effectors of transcription, and all three may respond to altered supercoiling at high temperature. Previous work indicated that thermal induction of TopA, an RNA polymerase-associated topoisomerase (Cheng et al 2003) is necessary for normal transcription patterns at high temperature and for thermal resistance (Qi et al 1996). HepA (RapA) is also an RNA polymerase-associated protein (Sukhodolets et al 2001) specifically required for RNA polymerase recycling of tightly compacted negatively supercoiled DNA.…”

groELExpression ingyrBMutants ofBorrelia burgdorferi

“…Therefore, the increase in GroEL is likely a response to DNA relaxation, which indicates that the groEL gene may be directly or indirectly regulated by DNA topology. DNA supercoiling is related to changes in topoisomerase levels (19). Reduced topoisomerase levels result in decreased DnaK and GroEL levels as well as attenuated thermotolerance in E. coli (19).…”

“…DNA supercoiling is related to changes in topoisomerase levels (19). Reduced topoisomerase levels result in decreased DnaK and GroEL levels as well as attenuated thermotolerance in E. coli (19). DNA gyrase inhibitors relax DNA supercoiling and increase synthesis of DnaK, another HSP (9).…”

Increased Sensitivity to Oxidative Challenges Associated with topA Deletion in Escherichia coli

“…Mutations in DNA topoisomerase I or gyrase genes have been shown to affect adaptation of bacterial organisms to high temperature (6,7,12,26,31), osmolarity (13,15,23), and availability of oxygen (33). There are at least two possible mechanisms by which topoisomerases can exert their influences (4, 31).…”

“…Primer extension experiments were carried out as previously described (26,27) to determine how NEM and hydrogen peroxide treatments affected the utilization of the topA promoters (Fig. 4).…”