Lactic acid bacteria (LAB) have a very long history of use in the manufacturing processes of fermented foods and a great deal of effort was made to investigate and manipulate the role of LAB in these processes. Today, the diverse group of LAB includes species that are among the best-studied microorganisms and proteolysis is one of the particular physiological traits of LAB of which detailed knowledge was obtained. The proteolytic system involved in casein utilization provides cells with essential amino acids during growth in milk and is also of industrial importance due to its contribution to the development of the organoleptic properties of fermented milk products. For the most extensively studied LAB, Lactococcus lactis, a model for casein proteolysis, transport, peptidolysis, and regulation thereof is now established. In addition to nutrient processing, cellular proteolysis plays a critical role in polypeptide quality control and in many regulatory circuits by keeping basal levels of regulatory proteins low and removing them when they are no longer needed. As part of the industrial processes, LAB are challenged by various stress conditions that are likely to affect metabolic activities, including proteolysis. While environmental stress responses of LAB have received increasing interest in recent years, our current knowledge on stress-related proteolysis in LAB is almost exclusively based on studies on L. lactis. This review provides the current status in the research of proteolytic systems of LAB with industrial relevance.

SUMMARYLactic acid bacteria (LAB) are important starter, commensal, or pathogenic microorganisms. The stress physiology of LAB has been studied in depth for over 2 decades, fueled mostly by the technological implications of LAB robustness in the food industry. Survival of probiotic LAB in the host and the potential relatedness of LAB virulence to their stress resilience have intensified interest in the field. Thus, a wealth of information concerning stress responses exists today for strains as diverse as starter (e.g.,Lactococcus lactis), probiotic (e.g., severalLactobacillusspp.), and pathogenic (e.g.,EnterococcusandStreptococcusspp.) LAB. Here we present the state of the art for LAB stress behavior. We describe the multitude of stresses that LAB are confronted with, and we present the experimental context used to study the stress responses of LAB, focusing on adaptation, habituation, and cross-protection as well as on self-induced multistress resistance in stationary phase, biofilms, and dormancy. We also consider stress responses at the population and single-cell levels. Subsequently, we concentrate on the stress defense mechanisms that have been reported to date, grouping them according to their direct participation in preserving cell energy, defending macromolecules, and protecting the cell envelope. Stress-induced responses of probiotic LAB and commensal/pathogenic LAB are highlighted separately due to the complexity of the peculiar multistress conditions to which these bacteria are subjected in their hosts. Induction of prophages under environmental stresses is then discussed. Finally, we present systems-based strategies to characterize the “stressome” of LAB and to engineer new food-related and probiotic LAB with improved stress tolerance.

SummaryClp proteolytic complexes consisting of a proteolytic core flanked by Clp ATPases are widely conserved in bacteria, and their biological roles have received considerable interest. In particular, mutants in the clp genes in the low-GC-content Gram-positive phyla Bacillales and Lactobacillales display a diverse range of phenotypic changes including general stress sensitivity, aberrant cell morphology, failure to initiate developmental programs, and for pathogens, severely attenuated virulence. Extensive research dedicated to unravelling the molecular mechanisms underlying these complex phenotypes has led to fascinating new insights that will be covered by this review. First, Clp ATPases and ClpPcontaining proteolytic complexes play indispensable roles in cellular protein quality control systems by refolding or degrading damaged proteins in both stressed and non-stressed cells. Secondly, ClpP proteases and the chaperone activity of Clp ATPases are important for controlling stability and activity of central transcriptional regulators, thereby exerting tremendous impact on cell physiology. Targets include major stress regulators like Spx (oxidative stress), the antisigma factor RsiW (alkaline stress) and HdiR (DNA damage) in addition to regulators of developmental programs like ComK (competence development), s H and Sda (sporulation). Thus, Clp proteins are central in co-ordinating developmental decisions and stress response in low GC Grampositive bacteria.

Lactobacillus rhamnosus GG (GG) is a widely used and intensively studied probiotic bacterium. Although the health benefits of strain GG are well documented, the systematic exploration of mechanisms by which this strain exerts probiotic effects in the host has only recently been initiated. The ability to survive the harsh conditions of the gastrointestinal tract, including gastric juice containing bile salts, is one of the vital characteristics that enables a probiotic bacterium to transiently colonize the host. Here we used gene expression profiling at the transcriptome and proteome levels to investigate the cellular response of strain GG toward bile under defined bioreactor conditions. The analyses revealed that in response to growth of strain GG in the presence of 0.2% ox gall the transcript levels of 316 genes changed significantly (p < 0.01, t test), and 42 proteins, including both intracellular and surface-exposed proteins (i.e. surfome), were differentially abundant (p < 0.01, t test in total proteome analysis; p < 0.05, t test in surfome analysis). Protein abundance changes correlated with transcriptome level changes for 14 of these proteins. The identified proteins suggest diverse and specific changes in general stress responses as well as in cell envelope-related functions, including in pathways affecting fatty acid composition, cell surface charge, and thickness of the exopolysaccharide layer. These changes are likely to strengthen the cell envelope against bile-induced stress and signal the GG cells of gut entrance. Notably, the surfome analyses demonstrated significant reduction in the abundance of a protein catalyzing the synthesis of exopolysaccharides, whereas a protein dedicated for active removal of bile compounds from the cells was up-regulated. These findings suggest a role for these proteins in facilitating the well founded interaction of strain GG with the host mucus in the presence of sublethal doses of bile. The significance of these findings in terms of the functionality of a probiotic bacterium is discussed. Molecular & Cellular

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