Hysteresis in myo-inositol utilization by Salmonella Typhimurium

Abstract

Growth of Salmonella enterica serovar Typhimurium strain 14028 with myo-inositol (MI) as the sole carbon and energy source is characterized by a bistable phenotype that manifests in a growth phenotype with an extraordinarily long and length-variable lag phase. However, in the presence of hydrogen carbonate, in the absence of IolR that represses the MI degradation pathway, or if cells are already adapted to minimal medium (MM) with MI, the lag phase is drastically shortened, and the bistable phenotype is abolished. We hypothesized that memory development or hysteresis is a further characteristic of MI degradation by S. Typhimurium; therefore, we investigated the transition from a short to a long lag phase in more detail. Growth experiments demonstrated that memory on the population level is successively lost within approximately 8 hr after cells, which had been adapted to MI utilization, were transferred to lysogeny broth (LB) medium. Flow cytometry (FC) analysis using a chromosomal fusion to P_iolE , a promoter controlling the expression of the enzymatic genes iolE and iolG involved in MI degradation, indicated a gradual reversion within a few hours from a population in the "ON" status with respect to iolE transcription to one that is mainly in the "OFF" status. Growth and FC experiments revealed that IolR does not affect hysteresis.

Keywords: Salmonella; hysteresis; metabolism; myo-inositol.

Figure 1

Hysteresis of myo‐inositol (MI)‐adapted S. Typhimurium. Strain 14028 was pre‐grown in minimal medium (MM) with MI, diluted 1:500 into lysogeny broth (LB) medium, and incubated for 4, 6, and 8 hr. An equal number of cells were then transferred into fresh MM with MI. MM is M9 medium supplemented with 2 mmol/L MgSO ₄, 0.1 mmol/L CaCl₂ supplemented with 55.5 mmol/L (1%, wt/vol) MI. Bacterial growth curves were obtained from cultures incubated at 37°C without agitation in 250 ml bulb flasks with 50 ml medium. OD ₆₀₀ was measured at the indicated time intervals. Standard deviations were calculated from triplicates

Figure 2

Flow cytometry (FC) analysis of MvP101 P_iolE::gfp. The reporter strain was pre‐grown for several passages in minimal medium (MM) with myo‐inositol (MI) and diluted 1:500 into 50 ml of fresh lysogeny broth (LB) medium. During growth, the OD ₆₀₀ was measured at the indicated time points, and samples were diluted in 1% phosphate‐buffered saline containing 2% formaldehyde to stabilize GFP (Bongaerts, Hautefort, Sidebotham, & Hinton, 2002). Fluorescence between 515 nm and 545 nm was measured with a FACS Aria I flow cytometer from Becton Dickinson at 488 nm excitation wavelength. A total of 10,000 events were monitored, and the collected data were analyzed using Flowing Software (v 2.5.1). The abscissa indicates the green fluorescence intensity in bi‐exponential scale, and the ordinate the number of bacteria in relation to the maximal cell counts. Each histogram represents the mean of three independent measurements. The insert illustrates the growth phase of the population during sample acquisition; the standard deviation was calculated from three triplicates. For all FC experiments, bacterial strains were grown overnight at 37°C in LB medium (Sambrook & Russell, 2001) or in MM with MI

Figure 3

Hysteresis of myo‐inositol (MI)‐adapted S. Typhimurium 14028 cells is IolR‐independent. (a) Growth curves of strain MvP101 ΔiolR. The in‐frame iolR (STM4417) deletion mutant of strain MvP101 was constructed by the one‐step method based on the phage λ Red recombinase (Datsenko & Wanner, 2000; Kröger & Fuchs, 2009); see Table  1 for plasmids and Table S1 for oligonucleotides. To obtain phage‐free colonies, bacteria were cultivated on green indicator plates (Provence & Curtiss, 1994). Experimental conditions were identical to those described in the legend of Figure 1. Growth curves are shown by dashed lines. (b) Flow cytometry (FC) analysis of MvP101 ΔiolR P_iolE::gfp. The fluorescence activity of P_iolE was monitored in the strain lacking iolR. The experiment was performed as described in the legend of Figure 2