Barotolerance is inducible by preincubation under hydrostatic pressure, cold-, osmotic- and acid-stress conditions in Lactobacillus sanfranciscensis DSM 20451T.

AIMS: This study addresses the inducibility of barotolerance by preincubation of Lactobacillus sanfranciscensis DSM 20451T under various sublethal stress conditions. METHODS AND RESULTS: Stress conditions which reduce the growth rate of L. sanfranciscensis DSM 20451T to 10% of its maximum were determined. These conditions were met at 43, 12.5 degrees C, a pH value of 3.7, 1.9% NaCl, or 80 MPa respectively. In contrast to heat preincubation, other prestresses, including salt, cold and pressure led to an increase of barotolerance by hydrostatic pressure of 300 MPa for 30 min. Stationary-phase cells also showed an increased barotolerance. Sublethal pressure leads to enhanced heat tolerance. CONCLUSIONS: Stress response to salt, low temperature and acidic pH as well as starvation overlap with that one to high pressure by inducing barotolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: Inactivation of bacteria by high pressure treatment is influenced by their history which modulates barotolerance. Mechanisms of barotolerance appear different from heat shock defence.

Characterization of the pressure-induced intermediate and unfolded state of red-shifted green fluorescent protein--a static and kinetic FTIR, UV/VIS and fluorescence spectroscopy study.

The green fluorescence proteins (GFP) are widely used as reporters in molecular and cell biology. For their use it in high-pressure microbiology and biotechnology studies, their structural properties, thermodynamic parameters and stability diagrams have to be known. We investigated the pressure stability of the red-shifted green fluorescent protein (rsGFP) using Fourier-transform infrared spectroscopy, fluorescence and UV/Vis spectroscopy. We found that rsGFP does not unfold up to approximately 9kbar at room temperature. Its unique three-dimensional structure is held responsible for the high-pressure stability. At higher temperatures, its secondary structure collapses below 9kbar (e.g. the denaturation pressure at 58 degrees C is 7.8kbar). The analysis of the IR data shows that the pressure-denatured state contains more disordered structures at the expense of a decrease of intramolecular beta-sheets. As indicated by the large volume change of DeltaV degrees (u) approximately -250(+/-50)mlmol(-1) at 58 degrees C, this highly cooperative transition can be interpreted as a collapse of the beta-can structure of rsGFP. For comparison, the temperature-induced unfolding of rsGFP has also been studied. At high temperature (T(m)=78 degrees C), the unfolding resulted in the formation of an aggregated state. Contrary to the pressure-induced unfolding, the temperature-induced unfolding and aggregation of GFP is irreversible. From the FT-IR data, a tentative p,T-stability diagram for the secondary structure collapse of GFP has been obtained. Furthermore, changes in fluorescence and absorptivity were found which are not correlated to the secondary structural changes. The fluorescence and UV/Vis data indicate smaller conformational changes in the chromophore region at much lower pressures ( approximately 4kbar) which are probably accompanied by the penetration of water into the beta-can structure. In order to investigate also the kinetics of this initial step, pressure-jump relaxation experiments were carried out. The partial activation volumes observed indicate that the conformational changes in the chromophore region when passing the transition state are indeed rather small, thus leading to a comparably small volume change of -20 ml mol(-1) only. The use of the chromophore absorption and fluorescence band of rsGFP in using GFP as reporter for gene expression and other microbiological studies under high pressure conditions is thus limited to pressures of about 4kbar, which still exceeds the pressure range relevant for studies in vivo in micro-organisms, including piezophilic bacteria from deep-sea environments.

In vitro stability and expression of green fluorescent protein under high pressure conditions.

AIMS: The objective of this work was to evaluate the use of wild-type GFP and mutant forms thereof as reporter for gene expression under high pressure conditions. METHODS AND RESULTS: The intensity of fluorescence after high pressure treatment was checked by subjecting cells, crude protein extracts containing GFPs and purified GFPs to pressures ranging from 100 MPa to 900 MPa. All tested GFP's retained fluorescence up to 600 MPa without loss of intensity. Expression of GFP under sublethal conditions was investigated in Escherichia coli with plasmid pQBI63, in which rsGFP is placed downstream of the T7 RNA polymerase binding site. T7 RNA polymerase is controlled in E. coli BL21 (DE3) pLysS by an IPTG inducible lacUV5 promoter. A pressure induced increase of GFP expression was monitored at 50 Mpa and 70 MPa. CONCLUSION: Fluorescence of GFPs is not influenced at pressures at which protein expression still occurs. We showed that the expression system used is inducible by pressurized conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proved GFP to be a suitable reporter for gene expression studies capable to detect pressure induced gene expression.