Role of sigma(B) in adaptation of Listeria monocytogenes to growth at low temperature

Abstract

The activity of sigma(B) in Listeria monocytogenes is stimulated by high osmolarity and is necessary for efficient uptake of osmoprotectants. Here we demonstrate that, during cold shock, sigma(B) contributes to adaptation in a growth phase-dependent manner and is necessary for efficient accumulation of betaine and carnitine as cryoprotectants.

FIG. 1

Primer extension analysis of the pattern of ς^B induction in L. monocytogenes after temperature downshift. Strain 10403S was grown in brain heart infusion to mid-logarithmic phase at 37°C followed by temperature downshift to 8°C. Growth was determined by measuring OD₆₀₀, and values obtained after cold shock are shown on the graph. Primer extension analysis was used to measure the activity of ς^B at the rsbV promoter. At times indicated by arrows on the graph, RNA was extracted from an aliquot of cells and 50 μg was used as a template for extension of the labeled VPROM2 primer (5). The numbers over the lanes in the autoradiograph correspond to the times indicated in the graph. A sequencing ladder generated with the same primer is shown to the left of the extension products.

FIG. 2

Cryoprotectant accumulation in 10403S and LMA2B at room temperature and immediately following temperature downshift. Cells were grown in DM at 37°C to mid-logarithmic phase (OD₆₀₀, ∼0.4), followed by immediate temperature downshift to 8°C. Prior to temperature shift and after acclimating for 5 min at 8°C, cell suspensions were collected and adjusted to approximately 0.12 mg of protein/ml. Uptake assays were performed at either 25°C (A) or 8°C (B) and started by addition of [¹⁴C]betaine or [³H]carnitine to each sample. Aliquots were removed over the time of the assay, harvested by centrifugation through oil, and counted by scintillation counting. Symbols: ■, 10403S plus [¹⁴C]betaine; ●, 10403S plus [³H]carnitine; □, LMA2B plus [¹⁴C]betaine; ○, LMA2B plus [³H]carnitine.

FIG. 3

Growth of 10403S and LMA2B at 8°C in the presence of cryoprotectants. Overnight stationary-phase cultures of 10403S and LMA2B growing in DM at 37°C were used to inoculate fresh DM supplemented with 1 mM betaine (A) or 1 mM carnitine (B) and incubated at 8°C. Symbols: ●, 10403S with no cryoprotectant; ○, LMA2B with no cryoprotectant; ■, 10403S with 1 mM betaine; □, LMA2B with 1 mM betaine; ⧫, 10403S with 1 mM carnitine; ◊, LMA2B with 1 mM carnitine.

FIG. 4

Cryoprotectant accumulation in 10403S and LMA2B growing at 8°C. Overnight stationary-phase cultures of 10403S and LMA2B growing in DM at 37°C were used to inoculate fresh DM followed by incubation at 8°C. Cells in mid-logarithmic growth (OD₆₀₀, ∼0.4) were collected, and cell suspensions were adjusted to approximately 0.12 mg of protein/ml. Uptake assays were performed at 8°C and started by addition of [¹⁴C]betaine or [³H]carnitine to each sample. Aliquots were removed over the time of the assay, harvested by centrifugation through oil, and counted by scintillation counting. Symbols: ■, 10403S plus [¹⁴C]betaine; ●, 10403S plus [³H]carnitine; □, LMA2B plus [¹⁴C]betaine; ○, LMA2B plus [³H]carnitine.