RESUMO
Salmonella enterica serovar Typhimurium (S. typhimurium) can cause food- and water-borne illness with diverse clinical manifestations. One key factor for S. typhimurium pathogenesis is the alternative sigma factor σE, which is encoded by the rpoE gene and controls the transcription of genes required for outer-membrane integrity in response to alterations in the bacterial envelope. The canonical pathway for σE activation involves proteolysis of the antisigma factor RseA, which is triggered by unfolded outer-membrane porins (OMPs) and lipopolysaccharides (LPS) that have accumulated in the periplasm. This study reports new stress factors that are able to activate σE expression. We demonstrate that UVA radiation induces σE activity in a pathway that is dependent on the stringent response regulator ppGpp. Survival assays revealed that rpoE has a role in the defence against lethal UVA doses that is mediated by functions that are dependent on and independent of the alternative sigma factor RpoS. We also report that the envelope stress generated by phage infection requires a functional rpoE gene for optimal bacterial tolerance and that it is able to induce σE activity in an RseA-dependent fashion. σE activity is also induced by hypo-osmotic shock in the absence of osmoregulated periplasmic glucans (OPGs). It is known that the rpoE gene is not essential in S. typhimurium. However, we report here two cases of the conditional lethality of rpoE mutations in this micro-organism. We demonstrate that rpoE mutations are not tolerated in the absence of OPGs (at low to moderate osmolarity) or LPS O-antigen. The latter case resembles that of the prototypic Escherichia coli strain K12, which neither synthesizes a complete LPS nor tolerates null rpoE mutations.
Assuntos
Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/fisiologia , Fator sigma/genética , Fator sigma/metabolismo , Estresse Fisiológico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófago P22/fisiologia , Glucanos/metabolismo , Guanosina Tetrafosfato/metabolismo , Viabilidade Microbiana , Mutação , Antígenos O/metabolismo , Pressão Osmótica , Salmonella typhimurium/efeitos da radiação , Salmonella typhimurium/virologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37 degrees C) through the use of urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterized an intermediate species, IU, which forms at 25 degrees C during unfolding or refolding of the portal protein in 2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On the other hand, at 1 degrees C, a two-state process is observed (DeltaGf = -2.2 kcal/mol). When subjected to HHP at 25 or 37 degrees C, the portal monomer undergoes partial denaturation, also forming an intermediate species, which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structure as seen by size-exclusion chromatography and electron microscopy. Again, at 1 degrees C the unfolding induced by HHP proved to be reversible, with DeltaGf = -2.4 kcal/mol and DeltaV = 72 mL/mol. Interestingly, at 25 degrees C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completely blocks its aggregation under HHP. These data are relevant to the process by which the portal protein assembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee the proper ring formation.
Assuntos
Bacteriófago P22/fisiologia , Dobramento de Proteína , Proteínas Virais/fisiologia , Cromatografia em Gel , Dicroísmo Circular , Relação Dose-Resposta a Droga , Pressão Hidrostática , Luz , Modelos Químicos , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos , Renaturação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Espalhamento de Radiação , Temperatura , Ureia/farmacologia , Proteínas Virais/química , Proteínas Virais/ultraestruturaRESUMO
This review describes the use of a simple genetic system that has provided important insight into the process of folding and, of its flipside, that of protein aggregation. These studies make use of the tail protein of the bacterial virus P22 which infects Salmonella typhimurium. This folding system serves as a model for a number protein structural elements and may also provide important insights into disease-related protein folding defects at a time when an increasing number of diseases are being shown to be due to protein folding alterations.