Effect of high temperature on viability of Lactobacillus casei and analysis of secreted and GroEL proteins profiles (original) (raw)
Related papers
Attributes of the Heat Shock Response in Three Species of Dairy Lactobacillus
Systematic and Applied Microbiology, 1997
Lactobacillus acidophilus, L. casei, and L. helveticus are industrially important bacteria for the manufacture of fermented dairy foods. Despite widespread commercial use, there is limited knowledge of basic physiological responses by these bacteria to dairy processing conditions. This study investigated the heat shock (HS) response in L. acidophilus NCFM, L. casei LC301 , and L. helveticus LH212. Thermotolerance experiments showed HS improved the ability of log phase L. acidophilus NCFM, L. casei LC301, and L. helveticus LH212 cells to withstand a 20 min high temperature incubation by approximately 27-, 5-and ll-fold, respectively. Two-dimensional polyacrylamide gel electrophoresis showed HS induced synthesis of several proteins in each Lactobacillus species, and Western blots revealed these molecules included homologs to the universally conversed heat shock proteins DnaK, GroEL, ClpB, and GrpE. DnaJ was also detected, but expression of this protein was not stimulated by HS in any of the Lactobacillus species tested.
Induction of Heat Shock Proteins DnaK, GroEL, and GroES by Salt Stress in Lactococcus lactis
1997
The bacterium Lactococcus lactis has become a model organism in studies of growth physiology and membrane transport, as a result of its simple fermentative metabolism. It is also used as a model for studying the importance of specific genes and functions during life in excess nutrients, by comparison of prototrophic wild-type strains and auxotrophic domesticated (dairy) strains. In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L. lactis subsp. cremoris laboratory strain MG1363, which was originally derived from a dairy strain. After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shock repressor, and the glycolytic enzymes pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase were identified by a combination of Western blotting and direct N-terminal amino acid sequencing of proteins from the gels. Of 400 to 500 visible proteins, 17 were induced more than twofold during heat stress. Two classes of heat stress proteins were identified from their temporal induction pattern. The fast-induced proteins (including DnaK) showed an abruptly increased rate of synthesis during the first 10 min, declining to intermediate levels after 15 min. GroEL and GroES, which also belong to this group, maintained a high rate of synthesis after 15 min. The class of slowly induced proteins exhibited a gradual increase in the rate of synthesis after the onset of stress. Unlike other organisms, all salt stress-induced proteins in L. lactis were also subjected to heat stress induction. DnaK, GroEL, and GroES showed similar temporal patterns of induction during salt stress, resembling the timing during heat stress although at a lower induction level. These data indicate an overlap between the heat shock and salt stress responses in L. lactis.
Analysis of heat shock gene expression in Lactococcus lactis MG1363
Microbiology, 1996
The induction of the heat shock response in Lactococcus lactis subsp. cremoris strain MG1363 was analysed at the RNA level using a novel RNA isolation procedure to prevent degradation. Cloning of the dnal and gm€L homologues was carried out. Northern blot analysis showed a similar induction pattern for dnaK, dnal and gm€LS after transfer from 30 "C to 43 "C when MG1363 was grown in defined medium. The dnaK gene showed a 100-fold induction level 15 min after temperature shifting. Induction of the first two genes in the dnaK operon, off7 and g @ , resembled the pattern observed for the above genes, although maximum induction was observed earlier for off7 and grlpE. Novel transcript sizes were detected in heat-shocked cells. The induction kinetics observed for fcsH suggested a different regulation for this gene. Experimental evidence for a pronounced transcriptional regulation being involved in the heat shock response in L. lactis MG1363 is presented. A gene located downstream of the dnaK operon in strain MG1363, named odd, was shown not to be regulated by heat shock.
Change in cell surface properties of Lactobacillus casei under heat shock treatment
We undertake this study in the aim to give new insight about the change in cellular physiological state under heat shock treatment and probiotic strain screening procedure. Different cell properties have been studied like adhesive ability to biotic and abiotic surfaces, the cell surface hydrophobicity and the fatty acids profiles. Compared to the normal cells, the heated cells increased their adhesive ability to biotic surface. However, the adhesion to abiotic surface was decreased. The cell surface hydrophobicity of the heated strains showed a significant decrease (P < 0.05). Our data revealed that high temperature change the fatty acids profiles of the treated cells, especially the proportions of unsaturated and saturated fatty acid. In fact, the ratio of saturated to unsaturated fatty acids of the heated Lactobacillus casei cells was significantly higher than that of the control cells (P < 0.05). The present finding could firstly add new insight about the response of probiotic to stressful conditions, such us the important role of cell membrane, considered as the first main structure to be damaged by physicochemical stress, in stress resistance because of their composition that can change in adaptation to harsh conditions. Secondly, there is no relationship between changes in membrane composition and fluidity induced by heat shock treatment and adhesion to biotic and abiotic surface.
Annals of Microbiology, 2012
We have investigated a potential link between small heat shock proteins (sHsps) and membranes in Lactobacillus plantarum WCFS1. The sHsp family is the best characterized protein subset among the L. plantarum Hsps, and it represents the principal heat shock regulon of Gram-positive bacteria in this bacterium. Exposure to benzylic alcohol (BA) (60 mM) alone was found to cause membrane fluidization in the absence of any other stress and to induce transcription of the heat shock genes (shs) to the same extent as a lethal heat shock. Using a fluorescence anisotropy-based method, we detected a reduction in the maximal fluidification level in two L. plantarum strains that singly over-produce Hsp18.5 and Hsp19.3. Overall, these results indicate that, under the stress conditions analysed, these two members of the sHsps family of L. plantarum WCFS1 have a membraneactivating effect. The findings also form a basis for proposing membrane fluidity as a new target for prestress treatments to enhance probiotic, food starter and bioproduction applications of L. plantarum.
Identification of sites of injury in Lactobacillus bulgaricus during heat stress
Journal of Applied Microbiology, 1997
Lactobacillus bulgaricus in skimmed milk at 62°, 64°, 65°and 66°C was studied. The response to increasing temperatures in this range was not linear, with temperatures at 65°C and above giving a lower survival rate than would be predicted from experiments at lower temperatures. To identify sites of injury at these temperatures, chemical markers were used. Heating at 64°C and below resulted in damage to the cytoplasmic membrane. At temperatures of 65°C and above chemical markers also indicated damage in the cell wall and proteins. Using differential scanning calorimetry analysis of whole cells of Lact. bulgaricus seven main peaks were observed (l-51, m 1 -61, m 2 -73, n-80, p-89, q-100, r-112°C). Three of these peaks (l r , m r and p r ) were the result of reversible reactions. Analysis of cell fractions identified the cell structure involved in giving rise to each of the three reversible peaks; l r , cell membrane lipids, m r , ribosomes, and p r , DNA. The evidence presented in this paper shows that irreversible reactions in the cell ribosomes are a critical site of damage in Lact. bulgaricus during heat stress in liquid media at 65°C and above.
Research in Microbiology, 2011
A small heat shock gene of Lactobacillus plantarum strain WCFS1 was deleted using a Cre-lox based system. Compared to the wild type, the Δhsp 18.55 mutant strain displayed a similar growth rate when cultivated either under optimal temperature or under different stress conditions such as heat, low pH and salt stress. However, a longer lag phase was observed when the Δhsp 18.55 mutant strain was cultivated under short intense heat stress (50 C). This suggests that the hsp 18.55 gene of L. plantarum may be involved in recovery of L. plantarum stressed cells in the early stage of high temperature stress. In addition, morphology of the mutant cells, investigated by scanning electron microscopy, revealed that cells clumped together and had rough surfaces, and that some of the cells had a shrunken empty appearance, which clearly contrasted with the characteristic rod-shaped, smooth-surface morphology of control L. plantarum cells. Furthermore, inactivation of the hsp 18.55 gene affected membrane fluidity and physicochemical surface properties of L. plantarum WCFS1.
Characterization of heat-shock proteins in Escherichia coli strains under thermal stress in vitro
Journal of Medical Microbiology, 2013
The aim of this study was to evaluate the effect of heat stress in in vitro conditions on the induction of heat-shock protein (Hsp)70 by Escherichia coli cells, and to determine the localization of Hsps in cell fractions. The material consisted of wild strains of E. coli isolated from the digestive tract of calves, suspended in an exponential-phase culture and subjected to 41.5 °C for 2 h. Individual fractions were analysed by SDS-PAGE and two-dimensional electrophoresis. Western blotting with mouse anti-Hsp70 and anti-Hsp60 mAbs was used to identify the proteins. Electrophoretic analysis of the heat-treated cells detected Hsp70 in all three fractions, cytoplasmic, periplasmic and membrane, which was confirmed by Western blotting. The proteins obtained had diverse localizations in the pH gradient in two-dimensional electrophoresis, which may indicate changes in their conformation and physical properties leading to stabilization and protection of intracellular structures in stress co...
PloS one, 2018
Prolonged heat stress is one of the harsh conditions Lactobacillus casei strains encounter as non-starter lactic acid bacteria in dairy product manufacture. To understand the physiological and molecular mechanisms through which Lb. casei GCRL163 adapts to persistent elevated temperature, label-free quantitative proteomics of cell-free extracts was used to characterize the global responses of the strain cultured anaerobically in bioreactors at 30 to 45°C, pH 6.5, together with GC-MS for fatty acid methyl ester analysis at different growth phases. At higher growth temperatures, repression of energy-consuming metabolic pathways, such as fatty acid, nucleotide and amino acid biosynthesis, was observed, while PTS- and ABC-type transporter systems associated with uptake of nitrogen and carbon sources were up-regulated. Alkaline shock protein Asp23_2 was only detected at 45°C, expressed at high abundance, and presumptive α-L-fucosidase only at 40 and 45°C, with highly increased abundance (...
FEMS Microbiology Letters, 2000
The mechanism of adaptation of bacteria to survive at elevated temperature in the human host and the expression of heat-shock proteins in response to stress was examined by labelling with [ 35S]methionine. An increase in culture temperature from 26°C to 37°C induced expression of certain bacterial proteins (70 and 60 kDa). Heat shock at 4O"C, cold shock (lOT), ethanol treatment or arsenite treatment also led to an increased expression of heat shock proteins of 70 and 60 kDa. Actinomycin D completely blocked the induction, indicating that transcription is required for the overexpression of stress proteins in Leuconostoc mesenteroides. N-terminal sequence analysis showed that these proteins were homologous to the highly conserved chaperone proteins DnaK and GroEL of Escherichia coli, respectively.