High binding affinity of repressor IolR avoids costs of untimely induction of myo-inositol utilization by Salmonella Typhimurium

Abstract

Growth of Salmonella enterica serovar Typhimurium strain 14028 with myo-inositol (MI) is characterized by a bistable phenotype that manifests with an extraordinarily long (34 h) and variable lag phase. When cells were pre-grown in minimal medium with MI, however, the lag phase shortened drastically to eight hours, and to six hours in the absence of the regulator IolR. To unravel the molecular mechanism behind this phenomenon, we investigated this repressor in more detail. Flow cytometry analysis of the iolR promoter at a single cell level demonstrated bistability of its transcriptional activation. Electrophoretic mobility shift assays were used to narrow the potential binding region of IolR and identified at least two binding sites in most iol gene promoters. Surface plasmon resonance spectroscopy quantified IolR binding and indicated its putative oligomerization and high binding affinity towards specific iol gene promoters. In competitive assays, the iolR deletion mutant, in which iol gene repression is abolished, showed a severe growth disadvantage of ~15% relative to the parental strain in rich medium. We hypothesize that the strong repression of iol gene transcription is required to maintain a balance between metabolic flexibility and fitness costs, which follow the inopportune induction of an unusual metabolic pathway.

Figure 1. Lag phases of S. Typhimurium during growth with MI.

(A) Strain 14028 pre-grown in MM/MI and then diluted 1:500 into MM/MI exhibited a much shorter lag phase in comparison with that of a culture inoculated with a pre-culture in LB medium. The growth curve of a culture taken from LB medium and re-inoculated into the same medium is shown as a control. (B) Lag phases were shortened by deletion of the repressor gene iolR. Growth curves of 14028 from (A) are displayed in black, and those of 14028 ∆iolR are indicated by dashed lines. Growth curves were derived as described in (A). Standard deviations were calculated from three biologically independent measurements.

Figure 2. Temporal FC analysis of P_iolR activity in MM with MI.

(A) MvP101 P_iolR::gfp or (B) MvP101 iolR::gfp grown in LB medium was adjusted to an OD₆₀₀ of 0.8, diluted 1:500 into MM/MI and incubated at 37 °C without agitation. At the indicated time points during growth, samples were collected and GFP-expressing cells were quantified by FC. The abscissa of each histogram represents the green fluorescence intensity at 515–545 nm on a bi-exponential scale, and the ordinate represents the numbers of bacteria relative to the maximal cell counts. Each histogram shows one representative data set of three biologically independent measurements. The inset illustrates the growth phase of the population during sample acquisition. (C) FC measurement of MvP101 P_rpsM::gfp as a control. The percentages of gfp-expressing cells (left) and non-fluorescent cells (right) are indicated. Samples were collected during a 48-h growth period after inoculation, and the average values and standard deviations of three independent growth experiments are shown; ***p < 0.001.

Figure 3. IolR binding to promoter regions within GEI4417/4436.

The genomic regions of the island are highlighted, and the different fragments used for EMSAs are shown. Black fragments were bound by IolR, grey fragments not. The molar excess of protein over DNA is indicated above the gels. The argS (100 ng) promoter fragment of served as a negative control and competitor (c) DNA, and the first lane in each EMSA was loaded without IolR. Asterisks indicate fragments selected for SPR spectroscopy.

Figure 4. SPR spectroscopy of IolR binding to P_iolT1, P_iolR and P_reiD.

The biotin-labeled DNA fragments P_iolT1 (A), P_iolR (B), and P_reiD (C) and control fragment P_pcfA (D) were captured on a streptavidin-coated sensor chip, and purified IolR was passed over the chip at a flow rate of 30 μl/min and temperature of 25 °C [concentrations of 0, 0.066, 0.165, 0.33, 0.66, 1.65, 3.3, 4.95, 6.6, 13.2, 19.8, 26.4, 33, 49.5, 66 (black and purple lines, internal reference), 165 and 330 nM] using a contact (association) time of 180 sec, followed by a 300-sec dissociation phase. The increase in RU correlates with an increasing IolR concentration. (E) IM analysis of the IolR-P_iolT1 interaction. The green and blue spots represent both interactions of IolR with the P_iolR DNA. The separate sensorgrams with the specific K_D values calculated from ON/OFF-rates map are shown in (F), together with the quantification of the in silico binding kinetics, e. g. the calculated association (k_a) and dissociation (k_d) rates as well as the quantitative portion of the total peak.

Figure 5. Competitive fitness assays of 14028 strains versus their iolR mutants.

Strains were grown separately in LB medium to an OD₆₀₀ of 0.5 and mixed together in a ratio of approximately 1:10 for 14028 vs. iolR::Kan^R (A) and 14028 dacB::Kan^R vs. ∆iolR (B). This mixture was used to inoculate 50 ml LB medium in an appropriate dilution of 1:250 to 1:1000. After a 24-h incubation, the cfu/ml were determined by plating culture samples on LB medium with or without kanamycin, and an aliquot of each culture was diluted 1:500 into fresh LB medium. This step was repeated twice. For each passage, the percentages of strains within the cultivated mixture are shown. (C) Competitive growth experiment with complemented strain 14028 iolR::kan^R/pBR-iolR against 14028/pBR322, and with strains 14028 and 14028 iolR::Kan^R Δ4418–4436; 14028 and 14028 iolR::Kan^R were used as a control. The inoculum was diluted 1:500. Cell numbers were calculated only after the third passage. Standard deviations were calculated from triplicate measurements of three independent experiments. Significance values were below 1% (p < 0.01*) or below 0.01% (p < 0,0001***).

Figure 6. Model of IolR interaction with its promoters.

Two different binding sites for IolR with high-affinity (A-site) and low-affinity (B-site) are present in the iol promoters exemplified here by P_reiD. (A) Initial binding of IolR dimers to the A-site of the promoter hampering RNA polymerase (RNAP) activity. (B) In case of higher copy numbers of IolR, also the B-site with lower affinity will be occupied by the repressor. (C) Oligomerization of IolR dimers result in trapping of the RNAP and completely inhibits reiD transcription. (D) Binding of the putative inducer (ligand) DKGP abolishes IolR binding, allowing the RNAP to start transcription. In this model, the binding or release of the B-site by IolR depends on the IolR copy numbers and the concentration of the inducer, leading to a pulse-like and highly reversible gene expression; together with the bistable autoregulation of iolR, the equilibrium between status (B) and (C) is decisive for iol gene transcription.