Project description:The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic, i.e. greater than additive, ability to inhibit the growth of Listeria monocytogenes. Full genome microarrays were used to investigate the synergistic transcriptomic response of two L. monocytogenes strains, h7858 (serotype 4b) and f6854 (serotype 1/2a), to organic acid, under conditions controlling for osmotic and cold stress. Strains were exposed to BHI broth at 7°C with 4.65% water phase (w.p.) NaCl at pH 6.1 treated with 2% w.p. potassium lactate, 0.14% w.p. sodium diacetate, the combination of both at the same levels, or no inhibitors as control. RNA was extracted 8h after exposure, during lag phase, to capture gene expression changes during adaptation to the organic acid stress. Treatment with organic acids induced massive global transcriptional changes, with 1041 and 640 genes differentially expressed in h7858 and f6854. Major effects of treatment with lactate and diacetate are (i) a total of 474 and 209 genes, for h7858 and f6854, that showed synergistic expression differences, (ii) differential expression of membrane ion transport genes including those encoding ABC transporters of metals and decreased multi-drug transporter expression many ABC, PTS, and drug transporter systems, including increased PTS sugar transport and decreased multi-drug transporter expression, and (iii) altered metabolism including induction of a nutrient limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of lactate and acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional synergies in L. monocytogenes growth inhibition could be achieved by treatments that interfere with cellular energy generation processes. The dye-swapped, single loop design hybridizes a single biological replicate of both 2% water phase lactate (PL) and 0.14% water phase acetate (SDA) treatments to both the control (CTRL) and combination (PLSDA) treatments (4 hybridizations), using opposite dye labels for each sample, and a second biological replicate is hybridized with dye assignments swapped (4 more hybridizations) to balance labeling effects. The design was repeated twice comprising 16 hybridizations over 4 biological replicates for each strain, and 32 total hybridizations over both h7858 and f6854.

Project description:A screening process was used to determine the aciduric capacities of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at pH 5.0 in the presence 4 different organic acids. Food and clinical isolates tended to be more resistant but strain genetic groupings were found to be only weakly linked to sodium diacetate (SDA)-resistance. Representative and comparatively aciduric food isolates FW04/0023 and FW04/0025 were found to accumulate reduced levels of acetate anion and K+ ion during growth in the presence of SDA, compared to more acid sensitive reference strains EGD (ATCC BAA-739) and ATCC 19111. The aciduric nature of FW04/0023 and FW04/0025 was also reflected by their comparatively high tolerance to pH 2.4 acid challenge; a property boosted by the presence of SDA, and growth-phase dependent acid tolerance. Transcriptomic analyses revealed increased levels of SDA did not activate or promulgate the response of any of the known acid protective mechanisms, except for acetoin biosynthesis. Elevated levels of unprotonated SDA (20 mM SDA at pH 5.0) was found to have broad effects on gene expression that could be differentiated from effects caused by mildly acidic conditions (pH 5.0) and substantial strain variation was also found. Collated SDA mainly effect genes associated with virulence, cell wall processes, DNA repair, and cofactor and lipid biosynthesis. Strain FW04/0025 was more responsive to elevated unprotonated SDA increasing the expression of 222 genes (>2-fold change, p<0.05), compared to 112 genes for strain EGD. Key differences between the strains in relation to SDA-enhanced transcript abundance in FW04/0023 were primarily associated with the cell wall, oxidative stress management, intermediary metabolism, and several hypothetical proteins. This complement of different responses could potentially alter phenotypes resulting in different cell envelope properties and metabolic properties that effect accumulation of acetate anion. The data demonstrates that amongst different L. monocytogenes strains acetate has differing effects that may ultimately influence growth efficiency under stressful conditions relevant to acidic foods and the gastrointestinal environment. The microarray component of the experiments had the aim of determining: i) To examine the affect of elevated levels of unprotonated sodium diacetate (21 mM sodium diacetate added to cultures at pH 5.0, resulting in ~7.7 mM unprotonated acetate) compared to mild acid stress at pH 5.0 in which lower levels of acetate accumulates through natural end-product formation. ii) To examine the gene expression responses of two L. monocytogenes strains that had different intrinsic acid resistances, including an organic acid resistant strain FW04/0025 and a more sensitive reference strain EGD on which genome the microarray oligonucleotide set is based. Two to four biologically replicated control cultures were labelled with Cy3 for each treatment sample with two sets of experimental conditions investigated for two different L. monocytogenes strains . Condition 1) â Mildly acidic stress: Control culture (Cy3-labelled RNA) - strains were grown to early- mid exponential growth phase at 25Â°C (in a shaking water bath) in brain-heart infusion broth at pH 7.3. Test cultures (Cy5-labelled RNA) â strains grown to early- mid exponential growth at 25Â°C phase in BHI broth adjusted to pH 5.0 (in a shaking water bath). Condition 2) â Organic acid (sodium diacetate) stress: Control culture (Cy3-labelled RNA) - strains were grown to exponential growth phase at 25Â°C (in a shaking water bath) in BHI broth at pH 5.0. Test cultures (Cy5-labelled RNA) â strains grown to exponential growth at 25Â°C phase in BHI broth adjusted to pH 5.0 and amended with 21 mM (0.3% w/v)sodium diacetate (in a shaking water bath).

Project description:A screening process was used to determine the aciduric capacities of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at pH 5.0 in the presence 4 different organic acids. Food and clinical isolates tended to be more resistant but strain genetic groupings were found to be only weakly linked to sodium diacetate (SDA)-resistance. Representative and comparatively aciduric food isolates FW04/0023 and FW04/0025 were found to accumulate reduced levels of acetate anion and K+ ion during growth in the presence of SDA, compared to more acid sensitive reference strains EGD (ATCC BAA-739) and ATCC 19111. The aciduric nature of FW04/0023 and FW04/0025 was also reflected by their comparatively high tolerance to pH 2.4 acid challenge; a property boosted by the presence of SDA, and growth-phase dependent acid tolerance. Transcriptomic analyses revealed increased levels of SDA did not activate or promulgate the response of any of the known acid protective mechanisms, except for acetoin biosynthesis. Elevated levels of unprotonated SDA (20 mM SDA at pH 5.0) was found to have broad effects on gene expression that could be differentiated from effects caused by mildly acidic conditions (pH 5.0) and substantial strain variation was also found. Collated SDA mainly effect genes associated with virulence, cell wall processes, DNA repair, and cofactor and lipid biosynthesis. Strain FW04/0025 was more responsive to elevated unprotonated SDA increasing the expression of 222 genes (>2-fold change, p<0.05), compared to 112 genes for strain EGD. Key differences between the strains in relation to SDA-enhanced transcript abundance in FW04/0023 were primarily associated with the cell wall, oxidative stress management, intermediary metabolism, and several hypothetical proteins. This complement of different responses could potentially alter phenotypes resulting in different cell envelope properties and metabolic properties that effect accumulation of acetate anion. The data demonstrates that amongst different L. monocytogenes strains acetate has differing effects that may ultimately influence growth efficiency under stressful conditions relevant to acidic foods and the gastrointestinal environment. The microarray component of the experiments had the aim of determining: i) To examine the affect of elevated levels of unprotonated sodium diacetate (21 mM sodium diacetate added to cultures at pH 5.0, resulting in ~7.7 mM unprotonated acetate) compared to mild acid stress at pH 5.0 in which lower levels of acetate accumulates through natural end-product formation. ii) To examine the gene expression responses of two L. monocytogenes strains that had different intrinsic acid resistances, including an organic acid resistant strain FW04/0025 and a more sensitive reference strain EGD on which genome the microarray oligonucleotide set is based.

Project description:PrfA activity was studied in L. monocytogenes strain EGD and in an isogenic prfA deletion mutant (EGDΔprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDΔprfApPrfA and EGDΔprfApPrfA*) after growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented either with one of the three PTS-carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type EGD strain in BHI and LB with either of these carbon sources, while PrfA activity was high in minimal medium in presence of glycerol but significantly reduced in presence of cellobiose. The strains expressing the prfA and prfA* gene under the prfA promoters, P1 and P2, produced equally large amounts of PrfA protein and high PrfA activity was observed in strain EGDΔprfApPrfA* under all growth conditions. In contrast, high PrfA activity in strain EGDΔprfApPrfA was only observed when this strain was cultured in BHI but not in LB or MM (in presence of either carbon source). A ptsH mutant (lacking a functional HPr) was able to grow in BHI suggesting that growth of L. monocytogenes in this culture medium is supported by carbon sources whose uptake and metabolism are independent of the PTS pathway. However, this mutant was unable to grow in LB and MM regardless which of the four carbon sources was added, suggesting that uptake of the used carbohydrates and the catabolism of glycerol depend fully on the functional common PTS pathway. Furthermore, the growth rates of L. monocytogenes are strongly reduced in presence of large amounts of PrfA protein when growing MM but less in LB and only slightly in BHI. The expression profiles of the genes encoding PTS permeases were determined in the three strains under various growth conditions. The data suggest that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases. This SuperSeries is composed of the following subset Series: GSE12143: Listeria monocytogenes EGD after growth BHI vs. LB vs. MM GSE12145: Listeria monocytogenes EGDΔprfApPrfA and EGDΔprfApPrfA* compared to the wild type strain EGD GSE12146: Listeria monocytogenes EGD and EGD-e

Project description:Background: Evolutionary engineering is a powerful approach to isolate suppressor mutants and industrially relevant genotypes. Until recently, DNA microarray analysis was the only affordable genome-wide approach to identify the responsible mutations. This situation has changed due to the rapidly decreasing costs of whole genome (re)sequencing. DNA microarray-based mRNA expression analysis and whole genome resequencing were combined in a study on lactate transport in Saccharomyces cerevisiae. Jen1p is the only S. cerevisiae lactate transporter reported in literature. To identify alternative lactate transporters, a jen1Δ strain was evolved for growth on lactate. Results: Two independent evolution experiments yielded Jen1p-independent growth on lactate (μmax 0.14 and 0.18 h-1 for single-cell lines IMW004 and IMW005, respectively). Whereas mRNA expression analysis did not provide leads, whole-genome resequencing showed different single nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Analysis of mRNA levels and depth of coverage of DNA sequencing combined with karyotyping, gene deletions and diagnostic PCR showed that in IMW004 an isochromosome III (~475 kb), which contains two additional copies of ADY2C755G, was formed via crossover between YCLWΔ15 and YCRCΔ6. Introduction of the ADY2 alleles in a jen1 ady2 strain resulted in growth on lactate (μmax 0.14 h-1 for Ady2pLeu219Val and 0.12 h-1 for Ady2pAla252Gly). Conclusions: Whole-genome resequencing of yeast strains obtained from independent evolution experiments enabled rapid identification of a key gene that was not identified by mRNA expression analysis of the same strains. Reverse metabolic engineering showed that mutated alleles of ADY2 (C655G and C755G) encode efficient lactate transporters. The goal of the present study was to investigate whether evolutionary engineering and subsequent elucidation of the underlying mutations can lead to the identification of alternative lactate transporters in S. cerevisiae. Transport of carboxylic acids plays a key role in weak organic acids stress and the responsible membrane transporters are often poorly studied and encoded by multiple redundant genes . Additionally, import of lactate is an essential step in the reutilization of lactate produced during intense exercise in mammals [8]. In S. cerevisiae, Jen1p was previously identified as the only efficient lactate importer by generation of a UV-mutant unable to grow on lactate and subsequent functional complementation with a genomic library . The lactate uptake rate of the resulting jen1 knockout strain was close to the detection limit. Interestingly, export of lactate in engineered lactate producing S. cerevisiae is unaffected by deletion of JEN1 (our unpublished results). These observations suggest the presence of at least one alternative lactate transporter. To test this, a jen1 knockout strain was constructed and evolved for faster growth on lactate as the sole carbon and energy source. The evolved strains were subjected to a combination of mRNA expression analysis and whole genome DNA (re)sequencing to identify the relevant mutations. The resulting leads were tested for lactate transport activity via knockout studies in the evolved strains and reverse engineering of the portable genetic elements into non-evolved strains.

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Transcriptional response of Listeria monocytogenes h7858 and f6854 to lactate and acetate

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