Increased expression of Mg ²⁺ transport proteins enhances the survival of Salmonella enterica at high temperature

Abstract

In a genetic analysis of the determinants of thermotolerance in S. enterica serovar Typhimurium, we isolated the chr-1 mutation that increased the resistance of exponential phase cells to killing by high temperature. This mutation is a single base change in the mgtA riboswitch that causes high-level constitutive expression of mgtA. We showed that another mgtA riboswitch mutation, ⌬UTR re-100 , which had been constructed by Cromie et al., also confers similar increased thermotolerance. Surprisingly, the chr-1 mutation is located at a position that would not be predicted to be important for the regulatory function of the riboswitch. We obtained physiological evidence suggesting that the chr-1 mutation increases the cytosolic free Mg 2؉ concentration. High-level expression of the heterologous MgtE Mg 2؉ transport protein of Bacillus subtilis also enhanced the thermotolerance of S. enterica. We hypothesize that increased Mg 2؉ accumulation might enhance thermotolerance by protecting the integrity of proteins or membranes, by mitigating oxidative damage or acting as an inducer of thermoprotective functions.heat tolerance ͉ riboswitch ͉ Mg 2ϩ homeostasis ͉ heat shock A s a pathogen that can grow in a free living state in various environments and in hosts ranging from ectothermic reptiles to warm-blooded mammals and birds whose temperature may be elevated by fever (1), Salmonella possesses sophisticated mechanisms for thermal adaptation. Salmonella is the most frequent causative agent of foodborne bacterial infections in the United States (2), and because heat treatment is the most common and economical means for inactivating food pathogens better understanding of the regulation of thermotolerance in this organism has important applications in food safety.The best-studied mechanism of thermal adaptation is the heat shock response. This response involves the transient induction of thermoprotective proteins by exposure to sublethal high temperatures, which enables organisms to survive subsequent treatments with higher temperatures that would be otherwise lethal (3). Thermotolerance can be regulated by a number of other factors, including stationary phase (4) and acidic or alkaline pH (5, 6).Another environmental condition that regulates thermoresistance is water activity (a w ). Exposure to elevated osmolality enhances the thermotolerance of bacteria in two ways: by elevating their upper limit of growth temperature (7,8) and increasing their viability at lethal high temperatures (9, 10). Our laboratory observed that the thermoprotective effect of high osmolality on the survival of Salmonella enterica serovar Typhimurium is reversed by glycine betaine (11). Glycine betaine is an osmoprotectant, which can alleviate the growth inhibitory effects of high osmolality (12). Along with ameliorating the osmotic inhibition of growth, this compound suppresses a number of other responses brought about by osmotic stress, such as the accumulation of K ϩ , the synthesis of trehalose, and the shrinkage of the cytoplasmic volume (13).To ...

Mg 2+ regulates transcription of mgtA in Salmonella Typhimurium via translation of proline codons during synthesis of the MgtL peptide

“…2). The same allele was previously obtained in an mgtA + background in the selection of mutations that confer enhanced thermotolerance (2). The elevation of transcription of mgtA by the C98T mutation and other premature nonsense mutations in mgtL (12) supports the notion that impaired translation of mgtL enables transcription to continue into the mgtA structural gene.…”

“…2), support the notion that the impaired translation in mgtL, due to stalling or +1-frameshifting, allows the formation of stem loop C, which sequesters the rut site within the mRNA secondary structure and precludes transcription termination by Rho. In addition, we isolated the C98T mutation in mgtL, which had been isolated previously by others (2,11) and was shown to result in premature translation termination (12). Despite the ability to form stem loop B conformation for Rho-dependent transcription termination, the C98T mutation in mgtL confers the constitutively ON phenotype (Fig.…”

“…Furthermore, Mg 2+ homeostasis is connected to thermotolerance in the food pathogen Salmonella enterica, because the survival of this organism at lethal high temperatures can be dramatically increased by the overproduction of Mg 2+ transport proteins (2). Salmonella has three uptake systems for Mg 2+ : MgtA, MgtB, and CorA.…”

Evaporation-induced stimulation of bacterial osmoregulation for electrical assessment of cell viability

“…These results demonstrate, for the first time, that activation of osmoregulatory transporters can be probed electrically through a label-free technique based on a microdevice (that can potentially be integrated in a massively parallel array). Finally, apart from the use as a sensor, the droplet-based, label-free platform is well-suited (as a complementary probe to traditional techniques) for answering a broad set of open biophysical questions, such as the hypothesis related to temperature-sensitive integrity of cell membrane (29). Fig.…”

“…As a reference, dead cells are prepared by heating an aliquot of live samples at 80°C for 20 min. In this case, we intentionally did not lyse the cells but just damaged their protein channels by heat to impair cells' ability to osmoregulate (29). Details of the sample preparation are given in SI Text, 2.…”