Folding and refolding of thermolabile and thermostable bacterial luciferases: the role of DnaKJ heat-shock proteins

AbstractBacterial luciferases are highly suitable test substrates for the analysis of refolding of misfolded proteins, as they are structurally labile and loose activity at 42°C. Heat-denatured thermolabile Vibrio fischeri luciferase and thermostable Photorhabdus luminescens luciferase were used as substrates. We found that their reactivation requires the activity of the DnaK chaperone system. The DnaKJ chaperones were dispensable in vivo for de novo folding at 30°C of the luciferase, but essential for refolding after a heat-shock. The rate and yield of DnaKJ refolding of the P. luminescens thermostable luciferase were to a marked degree lower as compared with the V. fischeri thermolabile luciferase. The refolding activity of the DnaKJ chaperones was examined at various temperatures. Between 30 and 37°C, the refolding rates of the V. fischeri luciferase decreased and the reaction reached a complete arrest at temperatures above 40°C. The rate of DnaKJ-mediated refolding of the thermostable luciferase at first increased between 30 and 37°C and then decreased at the range of 37–44°C. We observed that the rate of DnaKJ-mediated refolding of the heat-denatured P. luminescens thermostable luciferase, but not of the thermolabile V. fischeri luciferase, decreased during the prolonged incubation at a high (47°C) temperature. The efficiency and reversibility of protein refolding arrest during and after heat-shock strongly depended on the stability of the DnaKJ-denatured luciferase complex. It is supposed that the thermostable luciferase is released during the heat-shock, whereas the thermolabile luciferase remained bound to the chaperone

The Mode of Action of Cyclic Monoterpenes (−)-Limonene and (+)-α-Pinene on Bacterial Cells

A broad spectrum of volatile organic compounds’ (VOCs’) biological activities has attracted significant scientific interest, but their mechanisms of action remain little understood. The mechanism of action of two VOCs—the cyclic monoterpenes (−)-limonene and (+)-α-pinene—on bacteria was studied in this work. We used genetically engineered Escherichia coli bioluminescent strains harboring stress-responsive promoters (responsive to oxidative stress, DNA damage, SOS response, protein damage, heatshock, membrane damage) fused to the luxCDABE genes of Photorhabdus luminescens. We showed that (−)-limonene induces the PkatG and PsoxS promoters due to the formation of reactive oxygen species and, as a result, causes damage to DNA (SOSresponse), proteins (heat shock), and membrane (increases its permeability). The experimental data indicate that the action of (−)-limonene at high concentrations and prolonged incubation time makes degrading processes in cells irreversible. The effect of (+)-α-pinene is much weaker: it induces only heat shock in the bacteria. Moreover, we showed for the first time that (−)-limonene completely inhibits the DnaKJE–ClpB bichaperone-dependent refolding of heat-inactivated bacterial luciferase in both E. coli wild type and mutant ΔibpB strains. (+)-α-Pinene partially inhibits refolding only in ΔibpB mutant strain

The N-Terminal Domain of Aliivibrio fischeri LuxR Is a Target of the GroEL Chaperonin▿

Here we show that the C-terminal domain of LuxR activates the transcription of Aliivibrio fischeri luxICDABEG in Escherichia coli SKB178 gro+ and E. coli OFB1111 groEL673 strains to the same level. Using affinity chromatography, we showed that GroEL binds to the N-terminal domain of LuxR, pointing to a GroEL/GroES requirement for the folding of the N-terminal domain of LuxR

A Gene Encoding l-Methionine γ-Lyase Is Present in Enterobacteriaceae Family Genomes: Identification and Characterization of Citrobacter freundii l-Methionine γ-Lyase

Citrobacter freundii cells produce l-methionine γ-lyase when grown on a medium containing l-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded l-methionine γ-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3′-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found