High hydrostatic pressure (HHP) exerts diverse effects on microorganisms, leading to stress response and cell death. While inactivation of microorganisms by lethal HHP is well investigated in the context of food preservation and the hygienic safety of minimal food processes, sublethal HHP stress response and its effect on adaptation and cross-protection is less understood. In this study, the HHP stress response of Lactobacillus sanfranciscensis was characterized and compared with cold, heat, salt, acid and starvation stress at the proteome level by using 2-DE so as to provide insight into general versus specific stress responses. Sixteen proteins were found to be affected by HHP and were identified by using N-terminal amino acid sequencing and MS. Only one slightly increased protein was specific to the HHP response and showed homology to a clp protease. The other proteins were influenced by most of the investigated stresses in a similar way as HHP. The highest similarity in the HHP proteome was found to be with cold- and NaCl-stressed cells, with 11 overlapping proteins. At the proteome level, L. sanfranciscensis appears to use overlapping subsets of stress-inducible proteins rather than stereotype responses. Our data suggest that a specific pressure response does not exist in this bacteria.

Aims: This study addresses the inducibility of barotolerance by preincubation of Lactobacillus sanfranciscensis DSM 20451 T 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, 12AE5°C, a pH value of 3AE7, 1AE9% 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.

The effect of sublethal hydrostatic pressure on the transcriptome of Lactobacillus sanfranciscensis was determined using a shot-gun-microarray. Among the 750 spots that passed quality analysis 42 genes were induced, while six were repressed when cells were incubated at 45 MPa for 30 min. The nature of genes and their differential expression clearly indicate cellular efforts to counteract a decrease in translational capacity. The majority of high pressure affected genes were found to encode either translation factors (EF-G, EF-TU), ribosomal proteins (S2, L6, L11), genes changing translational accuracy or molecular chaperones (GroEL, ClpL). These data agree with previously reported effects observed in in vitro studies as well as with physiological and proteomic data. This study provides in vivo evidence to identify ribosomes and impaired translation among primary targets for high pressure treatment. The observed induction of heat as well as cold shock genes (e.g. hsp60, gyrA) may be explained as a result of high pressure affected protein synthesis.

Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7% of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2-to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK), while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

Incubation under sublethal high pressure (50 MPa) allowed the isolation of a piezotolerant mutant of Lactobacillus sanfranciscensis. Compared to the wild type this strain showed faster growth at 50 MPa and an altered temperature-dependent growth at ambient pressure. Additionally, an altered antibiotic resistance pattern was detected. To address the molecular basis of the mutation the genotypic characterisation was focused on alterations of ribosomal components. Northern analysis using ssrA (transfer mRNA) as probe revealed a constitutive overexpression in the mutant. A 2.2 fold induction after pressure shock and increased pressure sensitivity of a ssrA-insertional mutant of L. sanfranciscensis indicate the tmRNA as genetic determinant of a piezotolerance response in the wild type. Thus, we propose trans-translation and peptide tagging, processes that promote recycling of stalled ribosomes and prevent accumulation of abortively synthesised polypeptides to be involved in combating high-pressure damage and conferring moderate piezotolerance.