Timing of Induction of Osmotically Controlled Genes in Salmonella enterica Serovar Typhimurium, Determined with Quantitative Real-Time Reverse Transcription-PCR

Abstract

The signals that control the transcription of osmoregulated genes are not understood satisfactorily. The "turgor control model" suggested that the primary osmoregulatory signal in Enterobacteriaceae is turgor loss, which induces the kdp K ؉ transport operon and activates the Trk K ؉ permease. The ensuing increase in cytoplasmic K ؉ concentration was proposed to be the signal that turns on all secondary responses, including the induction of the proU (proline-glycine betaine transport) operon. The "ionic strength model" proposed that the regulatory signal for all osmotically controlled responses is the increase in the cytoplasmic ionic strength or macromolecular crowding after an osmotic upshift. The assumption in the turgor control model that the induction of kdp is a primary response to osmotic shock predicts that this response should precede all secondary responses. Both models predict that the induction of all osmotically activated responses should be independent of the chemical nature of the solute used to impose osmotic stress. We tested these predictions by quantitative real-time reverse transcription-PCR analysis of the expression of six osmotically regulated genes in Salmonella enterica serovar Typhimurium. After shock with 0.3 M NaCl, proU was induced at 4 min, proP and rpoS were induced at 4 to 6 min, kdp was induced at 8 to 9 min, and otsB and ompC were induced at 10 to 12 min. After an equivalent osmotic shock with 0.6 M sucrose, proU was induced with kinetics similar to those seen with NaCl, but induction of kdp was reduced 150-fold in comparison to induction by NaCl. Our results are inconsistent with both the turgor control and the ionic strength control models.Organisms respond to changes in external osmolality by accumulating or releasing low-molecular-weight "compatible solutes" that maintain the proper balance between internal and external osmolality. One of the prominent responses of Enterobacteriaceae to high ion concentrations is transcriptional induction a Ͼ100-fold of two operons: proU (proVWX), which encodes a transport system for the osmoprotectant compounds glycine betaine and proline, and kdpABC, which specifies the components of a high-affinity K ϩ transport system. The signal sensing mechanisms of the osmotic control of transcription of the kdp and proU operons are uncertain. Two osmoregulatory models were proposed for Enterobacteriaceae. The "turgor control model," which has been very influential in shaping thinking about bacterial osmoregulation, suggests that the fundamental signal is the loss of turgor resulting from the efflux of water after an osmotic upshift (3, 13). This model postulates that the first response of the cells to turgor loss is increased uptake of K ϩ , accomplished by stimulation of the activities of the constitutive, low-affinity K ϩ transport systems Trk and Kup and by transcriptional induction of the kdpABC operon. The model further proposes that elevated K ϩ acts as a second messenger to turn on all other osmotically activated responses, including the induct...

Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport

“…Proline accumulation has been shown to be an important strategy that bacteria use to cope with osmotic stress (Balaji et al, 2005;Grothe et al, 1986). The proU operon (encoding a transport system for proline and glycine betaine) promoter is induced by low water potential in Escherichia coli and Salmonella (Wright & Beattie, 2004).…”

Role of the alternative sigma factors σ E and σ S in survival of Salmonella enterica serovar Typhimurium during starvation, refrigeration and osmotic shock

“…Another stress relevant to food processing known to activate expression of both s E and s S dependent genes in E. coli and S. Typhimurium is hyperosmotic shock (HenggeAronis et al, 1993;Hengge-Aronis, 1996;Bianchi & Baneyx, 1999;Miticka et al, 2003;Balaji et al, 2005). However, again there has been little or no work on the role of s E and s S on the growth/survival of S. enterica in hyperosmotic environments.…”

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

“…into the surrounding medium without any damage to the cell envelope and/or cell lysis (10,22,23). In contrast, on osmotic upshift, another group of osmoregulatory transporters is activated to restore the natural turgor pressure (e.g., by uptaking solutes from the surrounding) (12,21,24). The bacterial response to different external osmotic pressures is summarized in SI Text, 1.…”

“…Under a microscope, bacteria cells are amazingly alive and perform a whole host of physiological functions, namely, multiplication through cell division, searching for resources by chemotaxis (5-9), controlling water pressure by exchange of ions (through the osmoregulatory system) (10)(11)(12), etc. However, since the introduction of the plate counting method almost 130 y ago, traditional viability assays, such as impedance microbiology, rely on cell multiplication to differentiate between dead and live cells (13)(14)(15)(16)(17)(18)(19).…”

“…The pressure difference (also known as turgor pressure) is defined by ΔΠ=Π cyt − Π out , where Π cyt and Π out are the cytoplasm and external osmotic pressures, respectively. The turgor pressure is regulated through activation of specific "emergency valves," which rapidly modulate the concentration of the solutes (including ionic species) in both the external and cytoplasmic solutions (10,12,20,21).…”