Kinetics of the thermal inactivation and the refolding of bacterial luciferases in Bacillus subtilis and in Escherichia coli differ

Eugeny Gnuchikh,Ilya Manukhov,Gennady Zavilgelsky,Ilyas Khaliullin,Vera Schukina,Ancha Baranova

doi:10.1371/journal.pone.0226576

Abstract

Here we present a study of the thermal inactivation and the refolding of the proteins in Gram positive Bacillus subtilis. To enable use of bacterial luciferases as the models for protein thermal inactivation and refolding in B. subtilis cells, we developed a variety of bright luminescent B. subtilis strains which express luxAB genes encoding luciferases of differing thermolability. The kinetics of the thermal inactivation and the refolding of luciferases from Photorhabdus luminescens and Photobacterium leiognathi were compared in Gram negative and Gram positive bacteria. In B. subtilis cells, these luciferases are substantially more thermostable than in Escherichia coli. Thermal inactivation of the thermostable luciferase P. luminescens in B. subtilis at 48.5°С behaves as a first-order reaction. In E.coli, the first order rate constant (Kt) of the thermal inactivation of luciferase in E. coli exceeds that observed in B. subtilis cells 2.9 times. Incubation time dependence curves for the thermal inactivation of the thermolabile luciferase of P. leiognathi luciferase in the cells of E. coli and B. subtilis may be described by first and third order kinetics, respectively. Here we shown that the levels and the rates of refolding of thermally inactivated luciferases in B. subtilis cells are substantially lower that that observed in E. coli. In dnaK-negative strains of B. subtilis, both the rates of thermal inactivation and the efficiency of refolding are similar to that observed in wild-type strains. These experiments point that the role that DnaKJE plays in thermostability of luciferases may be limited to bacterial species resembling E. coli.

Highlights

When exposed to mildly elevated temperatures, eukaryotic and prokaryotic thermolabile proteins transiently undergo partial or complete unfolding, resulting in a loss of their activity [1]
To quantify relative thermostability of luciferases in vivo, the luxAB gene expressing cells of B. subtilis 168 and E. coli BW25113 were grown at 28 ̊C and 37 ̊C for cells with luciferase from P. leiognathi and P. luminescens, respectively, until OD = 0.4–0.6 was reached
As opposed to E. coli, the cells of B. subtilis refold luciferases of P. leiognathi and P. luminescens to about the same levels, and with similar kinetics

Summary

Introduction

When exposed to mildly elevated temperatures, eukaryotic and prokaryotic thermolabile proteins transiently undergo partial or complete unfolding, resulting in a loss of their activity [1]. Eukaryotic and prokaryotic cells employ a variety of molecular chaperones, the most abundant and best. In studies of the folding, misfolding and refolding conditions in Escherichia coli, bacterial and firefly derived luciferases often serve as model substrates [10,11,12,13]. In present work we use the model bacterial luciferases differing in their thermostability to investigate the thermal inactivation and the refolding of the proteins in Gram positive B. subtilis. A variety of bright luminescent B. subtilis strains which express luxAB genes encoding luciferases from bacteria P. luminescens [14] and P. leiognathi [15], are utilized in the comparative study of the kinetics of the thermal inactivation and the refolding of the luciferases in Gram negative and Gram positive bacteria. We evaluated effects of dnaKJ genes on luciferase thermostability in cellular environments of B. subtilis and E. coli

Materials and methods

Results

Discussion