Alkali stress is an important means of inactivating undesirable pathogens in a wide range of situations, ranging from environmental cleaning of food processing environments, to the phagolysosomal killing of cells engulfed by mammalian phagocytes.
More...Alkali stress is an important means of inactivating undesirable pathogens in a wide range of situations, ranging from environmental cleaning of food processing environments, to the phagolysosomal killing of cells engulfed by mammalian phagocytes. Unfortunately, L. monocytogenes can launch an alkaline tolerance response (AlTR), significantly increasing persistence of the pathogen in such environments. This study compared the transcriptome patterns of alkali stressed and non alkali stressed L. monocytogenes 10403S cells, to elucidate the mechanisms by which this important pathogen adapts and/or grows during short or long-term alkali stress. Transcription profiles associated with alkali shock (AS) responses were obtained by DNA microarray analysis of mid-exponential cells suspended in pH 9 media for 15, 30 or 60 min. Transcription profiles associated with alkali adaptation (AA) were obtained by DNA microarray analysis of cells grown to mid-exponential phase in pH 9 media . Comparison of AS and AA transcription profiles with profiles from control (pH 7.0) cells identified over 2,000 genes that were differentially expressed under alkaline conditions. Rapid (15min) changes in expression included upregulation of genes encoding for multiple metabolic pathways, (including those associated with Na+/H+ antiporters), ABC transporters of functional compatible solutes such as carnitine, motility and virulence-associated genes as well as the σB controlled stress resistance network. Slower (30min and more) responses to AS and adaptation during growth in alkaline conditions (AA), included modest changes in mRNA concentrations, and genes involved in proton export.
Keywords: Time course study of gene expression response to alkaline shock and adaptation
Overall design: Overnight cultures of L. monocytogenes 10403S were inoculated (1:100) into 20 ml of BHI (pH 7) in a 50 ml Erlenmeyer flask, and incubated at 30°C with shaking at 200 rpm. Growth was monitored by measuring OD600, using a Beckman DU-65 spectrophotometer.
Cells to be subjected to alkali shock (AS) were harvested by centrifugation (8,000g x10min at 4°C) from 20 ml of this culture when the optical density at 600 nm reached 0.4. Recovered cells were washed, and immediately resuspended in 20 ml pre-warmed (30°C) pH 9 MBHI (test) or neutral MBHI (pH 7 control) for 15, 30 or 60 min.
Cells to be examined in terms of alkali adaptation (AA) were produced by inoculation of the overnight culture (1:100) into 20ml of MBHI adjusted at pH 9 (test) or pH 7 (control), and incubation at 30°C until they reached OD600 0.4.
In array analysis several controls were employed to minimise technical and biological variations, and ensure the quality of the data, i.e. i) each ORF was present in duplicate in each array, ii) each RNA preparation was used to make probes for at least two-separate arrays with reversal of incorporated dye and iii) three independent RNA batches from each condition were analysed. Only differences of ≥ 2 fold change in the levels of gene expression, which also had P values of 0.05, were recorded as significant.
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