Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor and DnaK

AbstractEscherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30°C. Here we show that the synthetic lethality of ΔtigΔdnaK52 cells is abrogated either by growth below 30°C or by overproduction of GroEL/GroES. At 23°C ΔtigΔdnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of ΔtigΔdnaK52 cells at 30°C and suppressed protein aggregation including proteins ≥60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates

Reappraisal of Vipera aspis Venom Neurotoxicity

BACKGROUND: The variation of venom composition with geography is an important aspect of intraspecific variability in the Vipera genus, although causes of this variability remain unclear. The diversity of snake venom is important both for our understanding of venomous snake evolution and for the preparation of relevant antivenoms to treat envenomations. A geographic intraspecific variation in snake venom composition was recently reported for Vipera aspis aspis venom in France. Since 1992, cases of human envenomation after Vipera aspis aspis bites in south-east France involving unexpected neurological signs were regularly reported. The presence of genes encoding PLA(2) neurotoxins in the Vaa snake genome led us to investigate any neurological symptom associated with snake bites in other regions of France and in neighboring countries. In parallel, we used several approaches to characterize the venom PLA(2) composition of the snakes captured in the same areas. [br/] METHODOLOGY/PRINCIPAL FINDINGS: We conducted an epidemiological survey of snake bites in various regions of France. In parallel, we carried out the analysis of the genes and the transcripts encoding venom PLA(2)s. We used SELDI technology to study the diversity of PLA(2) in various venom samples. Neurological signs (mainly cranial nerve disturbances) were reported after snake bites in three regions of France: Languedoc-Roussillon, Midi-Pyrénées and Provence-Alpes-Côte d'Azur. Genomes of Vipera aspis snakes from south-east France were shown to contain ammodytoxin isoforms never described in the genome of Vipera aspis from other French regions. Surprisingly, transcripts encoding venom neurotoxic PLA(2)s were found in snakes of Massif Central region. Accordingly, SELDI analysis of PLA(2) venom composition confirmed the existence of population of neurotoxic Vipera aspis snakes in the west part of the Massif Central mountains. [br/] CONCLUSIONS/SIGNIFICANCE: The association of epidemiological studies to genetic, biochemical and immunochemical analyses of snake venoms allowed a good evaluation of the potential neurotoxicity of snake bites. A correlation was found between the expression of neurological symptoms in humans and the intensity of the cross-reaction of venoms with anti-ammodytoxin antibodies, which is correlated with the level of neurotoxin (vaspin and/or ammodytoxin) expression in the venom. The origin of the two recently identified neurotoxic snake populations is discussed according to venom PLA(2) genome and transcriptome data

Functional Dissection of Escherichia coli Trigger Factor: Unraveling the Function of Individual Domains

In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of Δtig ΔdnaK in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, ΔtigΔdnaK cells expressing the N domain alone grew more slowly and showed less viability than ΔtigΔdnaK cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo