Samples were incubated at 37 °C for 20 min shaking (200 r.p.m.). Surviving cells were enumerated by serial dilution in PBS and subsequent plating onto BH agar. For colony forming unit (CFU) enumeration, overnight cultures (2 × 109 CFU mL−1) were washed twice in PBS and serially diluted to approximately 2 × 107 CFU mL−1. A further 1 Selleck AZD6738 in 10 dilution into the desired growth media was performed resulting in an inoculum of approximately 2 × 106 CFU mL−1. Counts were taken every 2 h over an 8 h period by serial dilution in PBS and enumeration on BHI agar. All agar plates were incubated
at 37 °C. For concurrently running OD600 nm readings, a sample was removed at the same time points and measured using a spectrophotometer. RNA extraction from stationary phase cells was carried out using the Macaloid method (Raya et al., 1998). The reverse transcriptase PCR was run using 4 μL random primer p(dN)6, 2 μL RNA, and 2 μL DEPC water (Sigma) at 65 °C for 10 min and put directly on ice. To these samples, 32 μL of a mastermix was added containing: 1 μL Expand Reverse Transcriptase (Roche), 8 μL 5× Buffer (Roche), 4 μL 100 mM dTT (Roche), 1 μL dNTP mix (dATP, dCTP, dGTP, dTTP – 10 mM), and 18 μL DEPC water. This reaction was run at 30 °C for 10 min, 42 °C for 3 h, and held at 4 °C. cDNA was confirmed through PCR using L142 and U141 primers and the wild-type
L. monocytogenes extracted DNA as a positive control. Quantitative real-time PCR was used for transcriptional analysis. The Universal Probe Library Assay Design Center (https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000)
was www.selleckchem.com/products/nivolumab.html used to design PCR primers that correspond to a specific probe in the library. Primer sequences, synthesized by MWG, and corresponding probes are listed in Online Resources (Table S1). The 16S rRNA gene was used as a housekeeping gene to compensate for any variability in the initial amount of Rho starting total RNA. Amplification reactions consisted of 2.5 μL of cDNA, 6.4 μL of 2× FastStart TaqMan Probe Master (Roche), primers (900 nM), and probe mix (250 nM). RNase-free water was added to bring the total volume of the reaction to 10 μL. Reactions were run in duplicate on 384-well plates using the LightCycler 480 System (Roche). Negative control reactions, without cDNA, were also included on the plate. Thermal cycling conditions were carried out according to manufacturer’s instructions (Roche), and the method (Livak & Schmittgen, 2001) was used to calculate the relative changes in gene expression from the qRT-PCR experiments. Zinc uptake systems have been described in numerous bacteria and include the high-affinity systems znuABC of E. coli and ycdHIycA of B. subtilis (Patzer & Hantke, 1998, 2000; Gaballa et al., 2002). For the most part, these systems are under the control of the zinc uptake regulator Zur, a homolog of which (ZurR) has been identified in L. monocytogenes (Dalet et al.