Unknown,Transcriptomics,Genomics,Proteomics

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Listeria monocytogenes Grown at Refrigeration Temperature Shows Reduced Acid Survival and an Altered Transcriptional Response to Acid Shock

ABSTRACT: Survival of the foodborne pathogen Listeria monocytogenes in acidic environments (e.g., stomach and low pH foods) is vital to its transmission. L. monocytogenes grows at temperatures as low as 2M-BM-0C, and refrigerated, ready-to-eat foods have been sources of L. monocytogenes outbreaks. The purpose of this study was to determine whether growth at a low temperature (i.e., 7M-BM-0C) affects the response of L. monocytogenes to sudden acid shock. A full genome microarray was used to determine changes in L. monocytogenes 10403S gene expression after exposure to acidified brain-heart infusion (BHI; pH 3.5) for 5 or 15 min. To determine changes in gene transcription after acid treatment, separate competitive hybridizations were performed between cDNA from untreated cells (grown at 7M-BM-0C or 37M-BM-0C to log or stationary phase) and (i) cells acid treated for 5 min or (ii) cells acid treated for 15 min. For L. monocytogenes grown to log or stationary phase, competitive hybridizations were performed between total cDNA from non-acid-treated cells grown to 7M-BM-0C and non-acid-treated cells grown to 37M-BM-0C to determine baseline differences in gene transcription between growth temperatures prior to acid treatment. For each experiment, four biological replications were completed. Hybridizations were carried out with dye swapping (i.e., for each comparison, each cDNA from each condition was labeled with each dye exactly twice) to help minimize dye incorporation bias.

ORGANISM(S): Listeria monocytogenes

SUBMITTER: Reid Ivy

PROVIDER: E-GEOD-22672 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organised quorum sensing (QS) systems via small acyl-homoserine lactone (AHL) signal molecules. Recently, a third bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the QS systems. To further dissect the various independent regulation levels for many QS induced genes and to evaluate the impact of PQS on the QS circuitry, we performed a transcriptome analysis of PAO1 cultures supplemented with PQS. The global transcriptional profile in response to PQS revealed a marked up-regulation of genes belonging to the tightly interdependent functional groups of iron acquisition and oxidative stress response. Remarkably, not only most of the differentially regulated genes but also the induction of a lacZ transcriptional fusion of rhlR could be traced back to a iron chelating effect of PQS. Nevertheless, although iron deficiency per se induced rhlR, there seems to be PQS specific effects that are independent of the PQS effect on P. aeruginosa iron homeostasis Experiment Overall Design: For RNA extraction bacteria were harvested at early logarithmic, late logarithmic and stationary phase of growth. Three independent cultures of PQS-treated and untreated PAO1 each were pooled and the RNA was immediately stabilized with RNAprotect Bacteria Reagent (Qiagen, Valencia, CA). RNA was isolated using the RNeasy kit (Qiagen), treated with DNaseI (Roche) for 30 min at 37M-BM-0C and re-purified with the RNeasy spin column. The subsequent steps of cDNA generation and Biotin-ddUTP terminal labelling were performed as described in the manufacturerM-bM-^@M-^Ys instructions for the P. aeruginosa GeneChipM-CM-^R. Fragmented and labelled cDNA was hybridised to a GeneChip at 50 M-BM-0C for 16 h. The washing steps, staining and scanning of the microarrays were performed using the Affymetrix GeneChip system. Data analysis was performed using the Affymetrix Microarray Suite Software 5.0 with Affymetrix default parameters. As expression analysis was performed in duplicate, a total of two GeneChips per culture condition was scanned at 570 nm, 3 M-BM-5m resolution in an Affymetrix GeneChip scanner. The signals were multiplied by a scaling factor to make the average signal for all the arrays equivalent. The data were imported into a Microsoft Access database capable of searching for genes, which were found in all four pairings defined by the Affymetrix Microarray Suite Software as having significant changes in their signal intensities. Data were combined with the latest annotation from the website of the P. aeruginosa PAO1 sequence and the community annotation project provided at www.pseudomonas.com.

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Listeria monocytogenes Grown at Refrigeration Temperature Shows Reduced Acid Survival and an Altered Transcriptional Response to Acid Shock

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