Validation of an internal control gene to apply reverse transcription quantitative PCR to study heat, cold and ethanol stresses in Lactobacillus plantarum (original) (raw)
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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.
Bioprocess and Biosystems Engineering, 2011
In this study, Lactobacillus rhamnosus, a renowned probiotic, was cultivated in fluctuating environment. Base gradients caused by a pH control in an industrial process and temperature gradients caused by uneven heating were simulated with a scale-down method. A pH gradient was created in a plug flow reactor (PFR). Expression of pH stress-related genes (atpA, aldB, cfa, groEL, hrcA and pstS) were studied as a relative gene expression study using ldhD as a reference gene. Expression measurements were carried out with the TRAC method. The responses of groEL, hrcA and atpA genes to temperature and pH changes were observed. The expression of phosphate uptake system-related pstS gene was induced almost linearly in the chemostat cultivation experiments when the base gradient in the PFR was increased. Correlations between the results from gene expression studies and freeze stability or acid stress survival were studied. However, by measuring the expression of these genes, we were not able to predict eventual freeze stability or survival from the acid stress test.
Improved adaptation to heat, cold, and solvent tolerance in Lactobacillus plantarum
Applied Microbiology and Biotechnology, 2007
The effect of overproducing each of the three small heat shock proteins (Hsp; Hsp 18.5, Hsp 18.55, and Hsp 19.3) was investigated in Lactobacillus plantarum strain WCFS1. Overproduction of the three genes, hsp 18.5, hsp 18.55, and hsp 19.3, translationally fused to the start codon of the ldhL gene yielded a protein of approximately 19 kDa, as estimated from Tricine sodium dodecyl sulfatepolyacrylamide gel electrophoresis in agreement with the predicted molecular weight of small Hsps. Small Hsp overproduction alleviated the reduction in growth rate triggered by exposing exponentially growing cells to heat shock (37 or 40°C) and cold shock (12°C). Moreover, overproduction of Hsp 18.55 and Hsp 19.3 led to an enhanced survival in the presence of butanol (1% v/v) or ethanol (12% v/v) treatment suggesting a potential role of L. plantarum small Hsps in solvent tolerance.
BMC Genomics
Background Psychrotrophic lactic acid bacteria (LAB) species are the dominant species in the microbiota of cold-stored modified-atmosphere-packaged food products and are the main cause of food spoilage. Despite the importance of psychrotrophic LAB, their response to cold or heat has not been studied. Here, we studied the transcriptome-level cold- and heat-shock response of spoilage lactic acid bacteria with time-series RNA-seq for Le. gelidum, Lc. piscium, and P. oligofermentans at 0 °C, 4 °C, 14 °C, 25 °C, and 28 °C. Results We observed that the cold-shock protein A (cspA) gene was the main cold-shock protein gene in all three species. Our results indicated that DEAD-box RNA helicase genes (cshA, cshB) also play a critical role in cold-shock response in psychrotrophic LAB. In addition, several RNase genes were involved in cold-shock response in Lc. piscium and P. oligofermentans. Moreover, gene network inference analysis provided candidate genes involved in cold-shock response. Rib...
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.
Quality Assurance and Safety of Crops & Foods, 2022
Lactic acid bacteria (LAB) are widespread in environments and can either have a positive impact because their ability to survive in harsh conditions and influence the product (probiotic properties, change of structure-EPS [exopolysaccharides], etc.), or a negative impact, (so not needed) because of their spoilage ability (beer, juices). High hydrostatic pressure (HHP), one of the non-thermal preservation methods used in the food industry, can force the LAB to activate the adaptative mechanisms. Under pressurization, the changes in the bacteria cells can occur at the transcriptional or translational level. This study evaluated the HHP on the single nucleotide polymorphism (SNP) changes in three genes, dnaK, ctsR, and hrcA, related to the stress-response mechanism in LAB. The correlation between the DNA polymorphism and the gene expression under HHP stress was assessed. The applied pressure of 300 MPa resulted in a low ratio of nonsynonymous substitutions to the synonymous substitutio...
2011
Abstract: Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) procedure was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3 % w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5 ' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidoph...
Journal of Bacteriology, 2000
An inverse PCR strategy based on degenerate primers has been used to identify new genes of the cold shock protein family in Lactobacillus plantarum. In addition to the two previously reported cspL and cspP genes, a third gene, cspC, has been cloned and characterized. All three genes encode small 66-amino-acid proteins with between 73 and 88% identity. Comparative Northern blot analyses showed that the level of cspL mRNA increases up to 17-fold after a temperature downshift, whereas the mRNA levels of cspC and cspP remain unchanged or increase only slightly (about two-to threefold). Cold induction of cspL mRNA is transient and delayed in time as a function of the severity of the temperature downshift. The cold shock behavior of the three csp mRNAs contrasts with that observed for four unrelated non-csp genes, which all showed a sharp decrease in mRNA level, followed in one case (bglH) by a progressive recovery of the transcript during prolonged cold exposure. Abundance of the three csp mRNAs was also found to vary during growth at optimal temperature (28°C). cspC and cspP mRNA levels are maximal during the lag period, whereas the abundance of the cspL transcript is highest during late-exponential-phase growth. The differential expression of the three L. plantarum csp genes can be related to sequence and structural differences in their untranslated regions. It also supports the view that the gene products fulfill separate and specific functions, under both cold shock and non-cold shock conditions.
Strain-Dependent Transcriptome Signatures for Robustness in Lactococcus lactis
PLOS ONE, 2016
Owing to their spoilage-preventing, texture-improving and flavor-enhancing properties, lactic acid bacteria have a long history of application in food fermentations . One of the most widely used lactic acid bacteria in the food industry is Lactococcus lactis, notably for the production of cheese and butter(milk) . These milk fermentation processes are typically initiated with the addition of starter cultures containing high concentrations of one or multiple L. lactis strains. During the production of these starter cultures prior to application in the food industry, L. lactis strains encounter severe stresses, for example heat and oxidative stress during spray drying . Although spray drying is a cost-effective and energy-efficient method for the preservation of starter cultures, it generally results in a relatively large decrease in viability as compared with other preservation methods such as freezing and freeze drying . Viability of starter cultures is essential for an adequate contribution to the fermentation endproduct, justifying the industrial interest to better understand and improve robustness .