Project description:Listeria monocytogenes is a ubiquitous and psychrophilic foodborne pathogen commonly found in raw materials, ready to eat products and food environments. It was previously demonstrated that L. monocytogenes can grow faster at low temperature when unsaturated fatty acids (UFA) are present in its environment. In this study, we used comparative gene expression profiling of RNA-sequencing data to understand the impact of UFA on the behavior and cold adaptation of L. monocytogenes. We demonstrate that the incorporation of UFA into the membrane induces changes in the regulation of overall fatty acid biosynthesis, which prompts us to propose two hypotheses for UFA synthesis in L. monocytogenes. The general stress response is also highly impacted by the incorporation of UFA into the membrane at low temperature. In particular, we hypothesize that transcriptional regulation of cspB is not a temperature dependent mechanism, but could be related to a membrane fluidity stimulus. Furthermore, when UFA are incorporated into the membrane at low temperature, we observed overexpression of genes involved in flagella assembly. This study sheds light on the cold adaptation of L. monocytogenes in the presence of exogenous FA and on potential concerns for controlling these bacteria in food environments.
Project description:The aim of this work is to investigate whether A. thaliana senses low temperature by perceiving changes in membrane fluidity. To this end, we have performed an experiment to test whether mutant or transgenic plants with altered membrane lipid composition, regulate their gene expression in the same manner as wild type plants in response to cold. Previous work has demonstrated that a change in the expression levels of a number of genes is important in acquiring tolerance to low temperatures. Chemicals which rigidify cell membranes in such a way as to mimic the effects of cold have been shown to be able to induce the expression of such genes. However, because of the non-specific nature of such chemical treatments, it has not been possible to demonstrate unequivocally that the changes in gene expression observed were the result of changes in membrane fluidity. All of the mutants used in our experiment, fab1, fad2-2 and the fad3/fad7/fad8 mutant, have increased lipid saturation levels compared to wild type plants and are thought to have reduced membrane fluidity. The fab1 mutant is also known to be sensitive to chilling. In the fab1 mutant the elongation of 16:0 fatty acids to 18:0 is reduced. The fad2-2 mutant has reduced 18:1 desaturase activity and hence reduced amounts of polyunsaturated phospholipids. The fad3/fad7/fad8 triple mutant is deficient in 18:2 desaturase activity and consequently unable to synthesise trienoic fatty acids. The transgenic line used contained a 35S::FAD3 transgene and in contrast to the mutants tested, should have increased lipid desaturation and increased membrane fluidity. A. thaliana ecotype Col-0 was used as the wild type control for the fab1 and fad2-2 mutants, in addition to the 35S::FAD3 line. The fad3/fad7/fad8 mutant had previously been transformed with the 35S::apoaequorin transgene and a Columbia line expressing apoaequorin under the control of the same promoter, was included to control for the presence of aequorin. Nine day old seedlings grown in petri-dishes on MS were transferred from their growth room (20 oC, 16 h photoperiod, 100 E m-2 s-1) to a growth cabinet (20 oC, 16 h photoperiod,160 m-2 s-1) 24 hours before the experiment began. The next day, one petri-dish of seedlings from each line of plants used was transferred to a cabinet running at 5 oC (16 h photoperiod,160 E m-2 s-1). Control plates remained at 20 oC. Seedlings were harvested after three hours and frozen in liquid nitrogen.
Project description:Several lipocalin genes from higher plants were shown to be responsive to both high and low temperature stresses and have been named as temperature-induced lipocalin (Til). In this study, a reverse genetic approach was taken to elucidate the role of Arabidopsis Til1 (At5g58070) in thermotolerance. We showed that Til1 proteins was constitutively expressed and increased significantly after heat shock treatment. A T-DNA knockout line of Til1, designated as til1-1, could not produce Til1 and showed severe defects in basal and acquired thermotolerance. Introducing a wild type copy of Til1 gene into til1-1 complemented the mutant phenotype. Over-expression of Til1 in the wild type plant did not enhance thermotolerance. Til1 is peripherally associated with plasma membrane, suggesting a regulatory or protective role of this protein in membrane function. Transcriptomic analysis showed that the heat shock response in til1-1 was not altered as compared to the wild type plants. The temperature threshold for heat shock protein induction was not affected by the level of Til1. Ion leakage analysis revealed no significant difference in membrane stability between the wild type and til1-1 seedlings. These results suggested that Til1 is not involved in regulating membrane fluidity or stability. Nevertheless, the level of malondialdehyde was significantly higher in til1-1 than in the wild type after severe heat treatment. The mutant plants were also more sensitive than the wild type to tert-butyl hydroperoxide, a reagent that induces lipid peroxidation. Taken together, our data indicate that Til1 is an essential component for thermotolerance probably by acting against lipid peroxidation induced by severe heat stress. Experiment Overall Design: Total RNA was isolated from the seedlings of 7-d old wild-type and til1-1 mutant seedlings (a pool of about 100 plants per treatment in duplicates) harvested immediately after heat shock treatment. In this experiment, total 8 chips were used, 1 each for 2 biological replicates of the control and HS-treated samples for the wild type and mutant plants.
Project description:Five SAGE libraries were generated from A. thaliana leaf tissue collected at time points ranging from 30 minutes to one week of low temperature treatment (4°C). Over 240,000 high quality SAGE tags, corresponding to 16,629 annotated genes, provided a comprehensive survey of changes in the transcriptome in response to low temperature, from perception of the stress to acquisition of freezing tolerance. Keywords: SAGE; time course; stress response; cold acclimation; freezing tolerance
Project description:All free-living microorganisms homeostatically maintain the fluidity of their membranes by adapting lipid composition to environmental temperatures. A quantitative description of how organisms maintain constant fluidity at all growth temperatures has not been achieved. By quantifying both enzymes and metabolic intermediates of the Escherichia coli fatty acid and phospholipid synthesis pathways, we discover how E. coli measures steady-state temperature and restores optimal membrane fluidity within a single generation after temperature shocks.
Project description:The aim of this work is to investigate whether A. thaliana senses low temperature by perceiving changes in membrane fluidity. To this end, we have performed an experiment to test whether mutant or transgenic plants with altered membrane lipid composition, regulate their gene expression in the same manner as wild type plants in response to cold. Previous work has demonstrated that a change in the expression levels of a number of genes is important in acquiring tolerance to low temperatures. Chemicals which rigidify cell membranes in such a way as to mimic the effects of cold have been shown to be able to induce the expression of such genes. However, because of the non-specific nature of such chemical treatments, it has not been possible to demonstrate unequivocally that the changes in gene expression observed were the result of changes in membrane fluidity. All of the mutants used in our experiment, fab1, fad2-2 and the fad3/fad7/fad8 mutant, have increased lipid saturation levels compared to wild type plants and are thought to have reduced membrane fluidity. The fab1 mutant is also known to be sensitive to chilling. In the fab1 mutant the elongation of 16:0 fatty acids to 18:0 is reduced. The fad2-2 mutant has reduced 18:1 desaturase activity and hence reduced amounts of polyunsaturated phospholipids. The fad3/fad7/fad8 triple mutant is deficient in 18:2 desaturase activity and consequently unable to synthesise trienoic fatty acids. The transgenic line used contained a 35S::FAD3 transgene and in contrast to the mutants tested, should have increased lipid desaturation and increased membrane fluidity. A. thaliana ecotype Col-0 was used as the wild type control for the fab1 and fad2-2 mutants, in addition to the 35S::FAD3 line. The fad3/fad7/fad8 mutant had previously been transformed with the 35S::apoaequorin transgene and a Columbia line expressing apoaequorin under the control of the same promoter, was included to control for the presence of aequorin. Nine day old seedlings grown in petri-dishes on MS were transferred from their growth room (20 oC, 16 h photoperiod, 100 E m-2 s-1) to a growth cabinet (20 oC, 16 h photoperiod,160 m-2 s-1) 24 hours before the experiment began. The next day, one petri-dish of seedlings from each line of plants used was transferred to a cabinet running at 5 oC (16 h photoperiod,160 E m-2 s-1). Control plates remained at 20 oC. Seedlings were harvested after three hours and frozen in liquid nitrogen. 12 samples were used in this experiment
Project description:How plants control the transition to flowering in response to ambient temperature is only beginning to be understood. In Arabidopsis thaliana, the MADS-box transcription factor genes FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) have key roles in this process. FLM is subject to temperature-dependent alternative splicing, producing two splice variants, FLM-β and FLM-δ, which compete for interaction with the floral repressor SVP. The SVP/FLM-β complex is predominately formed at low temperatures and prevents precocious flowering. In contrast, the competing SVP FLM-δ complex is impaired in DNA binding and acts as a dominant negative activator of flowering at higher temperatures. Our results demonstrate the importance of temperature-dependent alternative splicing in modulating the timing of the floral transition in response to environmental change.