All major goals of the tasks have been achieved. In some areas unexpected additional results have been achieved, which highlight high pressure as a powerful tool in the investigation of macromolecular interaction.
Tasks 1 and 2
Characterization of HHP responses on the gene/genome/transcriptome and on the enzyme/proteome level
Bacterial systems: Proteomic and transcriptomic approaches and genetic analyses of high pressure mutants revealed that high pressure is a powerful tool to investigate cellular processes far beyond the mere inactivation of bacteria. Two approaches can be separated for which different responses to high pressure are apparent. Organisms can be shocked by short (nearly) lethal pressure puls(es) (e.g.150-400 MPa) and their response is characterized upon recovery. Alternatively, bacteria can be grown under sublethal (comparably low) pressures (e.g. 20-150 MPa) for a prolonged time possibly resulting in adaptation and (another type of) mutation. Two major effectors of the pressure response were identified in the different approaches. Overexpression of ssrA enables growth under high pressure by helping to maintain ribosomal function in Gram-positive bacteria (Lactobacillus sanfranciscensis). The restriction endonuclease mrr, specifically cleaving methylated DNA was identified as a major mediator of the high pressure response in Gram-negatives (E. coli).
Primary thermodynamic targets present in the cell were identified to be the cellular membrane and the ribosome. It appears that translation is stalled in vivo at pressures above 40 MPa. Ribosomes can be returned to functionality by trans-translation and peptide tagging mediated by tmRNA and the ClpX protease/chaperon system. Both systems are turned on as a specific reaction of high pressure stress. Many other stress responses observed appear to be secondary effects, which are the result of the typical response pathways present in a respective cell. As the approaches in E. coli and L. sanfranciscensis are highly complementary, the respective systems were studied in the respective other system in close collaboration of the groups in Munich, Germany and Leuven, Belgium. Furthermore, it was shown that the expression of virulence factors and changes in antibiotic resistance can be triggered by high pressure treatments. This was studied in collaboration of the groups in Munich, Germany and Monells, Spain.
Eukaryotic systems: Systems involving muscle cells and chondrocytes are investigated in this WG. It was demonstrated that these systems also as a result of their larger size as compared to bacteria, are valuable models in using pressure as a tool in cell biology. High pressure microscopy facilities at the Technische Universität München have already been used to study muscle cells and their ion channels under pressure in situ in an STSM (self funded within Germany). These investigations were continued in an STSM between the Heidelberg group (Germany) and the JAMSTEC (Japan). For the first time resting membrane potentials recorded on-site in intact skeletal muscles from deep sea fish (gonostoma gracilis sp.) salvaged from depths up to 1.000 m were recorded. In ‘in situ’ high pressure microscopy it was shown that intracellular Ca2+ homeostasis is pressure-dependently altered in single muscle cells. Ca2+ leaks from intracellular stores upon compression and is first taken up and buffered by mitochondria. Then, at about 30 MPa, mitochondria buffer capacity breaks down and Ca2+ ions are poured back into the cytosol resulting in irreversible contracture of the cells. For these experiments, a high pressure optical vessel was combined with a confocal laser scanning microscope. The results are interpreted in terms of a model for high pressure limits in mammalian muscle for prolonged high pressure applications. There is a rather sharp limit for functional breakdown at ~25 MPa that coincides with the deepest dive depths recorded for mammals (sperm whales). Therefore, deep sea fish must have evolved some protective mechanisms of the membrane level to keep up their excitability. In these fish depth and temperature both alter passive membrane parameters with a tendency towards larger relative Na+ permeabilities and intracellular K+ concentrations in samples from deeper depths (e.g. 700-800 m vs. 200-400 m).
Functional analysis of cellular membranes and membrane proteins
In a joint project of the German research foundation members of WG007 (TUM, Germany) collaborated with a member of WG006 (Universität Dortmund, Germany) on the piezophysiology of bacterial membranes. Systems were developed to study membrane protein interaction in vivo and in membrane vesicles. It was shown, that high pressure can mediate signaling processes by interfering with protein dimerization in a cellular membrane system. Also, membrane transporters are affected via their changes ATPase activity. The systems involved the multi-drug-transporter LmrA of Lactococcus lactis and the ToxRS systems of Vibrio cholerae and the deep sea bacterium Photobacterium profundum. An STSM is ongoing until the end of the action between Reading and Munich to study protein secretion as a function of high pressure. All of the proteins released from pressure-treated cells had a molecular weight below 80 kDa. This cut-off point is consistent with the suggestion that the peptidoglycan of the cell wall acts as a molecular sieve for proteins leaking from bacterial cells.
Tasks 4 and 5
Characterization of HHP responses on the population level
Interaction of HHP effects on microorganisms with environmental factors
The characterisation of responses to high pressure of microorganisms in food systems has been elucidated with respect to population level and interaction of high pressure with environmental parameters in the groups situated in Belfast, UK, Reading UK and Monells, Spain. Mathematical models were established to describe the inactivation of bacteria in these systems. A new primary model for inactivation of microbes under pressure was constructed to describe non-log-linear inactivation kinetics of pressure-treated bacteria. The model assumes a first-order process in which the specific inactivation rate changes inversely with the square root of time. The model gave reasonable fits to experimental data over six to seven orders of magnitude. It was also tested on 138 published data sets and provided good fits in about 70% of cases in which the shape of the curve followed the typical convex upward form. In the remainder of published examples, The model parameters varied regularly with pressure which may reflect a genuine mechanistic basis for the model. This property also allowed the calculation of (a) parameters analogous to D and z in thermal processing, and (b) the apparent thermodynamic volumes of activation associated with the lethal events. Further, application of fuzzy logic and neuronal networks provided by the Munich group was successfully applied to describe metabolic and cellular functions under high pressure. With these it was possible to include into the models intermediate states of inactivation and interaction with the matrix. Synergisms with antimicrobial compounds and high pressure were studied in Monells, Spain and Leuven. Belgium.
The WG has held 3 meetings, 2004 in Munich, Germany, 2005 in Reading, UK and along the EHPRG symposium 4-6th September 2006 in Prague, CZ as a joint meeting of WGs 6 and 7.
Three STSMs Spain => Germany, Germany => Japan, and UK => Germany were successfully performed in 2006 and 2007 on the expression of virulence factors in Enterococcus faecalis, the investigation on muscle cells of deep sea eels, and the high pressure induced protein release in E. coli, respectively. The STSM Germany => Japan involved our Non-EU member offering the unique opportunity of a deep sea cruise and experiments with cells of deeps sea eels.
Interactions with other WGs:
In a joint project of the German research foundation members of WG007 (TUM, Germany) collaborated with a member of WG006 (Universität Dortmund, Germany) on the piezophysiology of bacterial membranes. Another highlight was the joined WG meeting in Prague. Some of the scientists in WG007 will participate in the final meeting in Bordeaux in October 2007 to share their results and finish joint publications from ongoing collaborations.
The first book on “High pressure microbiology” with several members of the WG being authors, will be printed in 2008 by ASM press. This is considered a major achieved in jointly publishing the results of the WG007 during COST and beyond.