The regulation of leuO transcription by BglJ-RcsB and LeuO is an important element in the control of the LeuO master regulator. To address regulation of leuO transcription that is directed by at least two promoters (P_leuO) in dependence of the concentrations of BglJ and LeuO, a suitable experimental system was established. First, the mVenus reporter gene (coding for the yellow fluorescent protein mVenus) was fused to the leuO promoter-regulatory region by replacement of the leuO gene resulting in allele P_leuO mVenus, ΔleuO (Figure 1B). Second, BglJ and LeuO were ectopically expressed from two different sets of plasmids. In one plasmid set, bglJ was expressed under control of the IPTG-inducible lacUV5 promoter (P_UV5) using low-copy plasmid pKETS26 (pSC origin of replication), and leuO was expressed under control of the arabinose-inducible P_BAD promoter using the low to medium copy plasmid pKES303 (pBAD30-derived, p15A origin of replication). In the other plasmid set, bglJ was expressed under control of the P_BAD promoter (pKES302, p15A-ori) and leuO under control of IPTG-inducible P_tac promoter (pKEHB27, pSC-ori). The genes encoding the AraC and the LacI regulators, respectively, are also carried on these plasmids. Additionally, the yjjQ-bglJ operon was deleted resulting in allele Δ(yjjP-yjjQ-bglJ) to ensure that only plasmid-encoded BglJ is present in the cell. Note that RcsB is not limiting for activation of leuO and other loci by BglJ-RcsB (Salscheider et al., 2014; Pannen et al., 2016). Third, to allow controlled and finely tunable expression of bglJ and leuO directed by the arabinose-inducible P_BAD promoter and the IPTG-inducible P_UV5 and P_tac promoters, respectively, additional mutations and modifications were introduced into the reporter strain (Figure 1B). The P_UV5 promoter is gradually induced over a range of inducer concentrations (IPTG) when the lactose permease gene lacY is deleted (Jensen et al., 1993). Therefore, the lacZYA operon and the lacI gene were deleted in the reporter strain resulting in allele Δ(lacI-lacZYA) (Table 1). Likewise, the arabinose regulon was modified to ensure a gradual induction of the P_BAD promoter with arabinose, as described before (Khlebnikov et al., 2001; Kogenaru and Tans, 2014). Briefly, the P_BAD promoter is known to have a stochastic behavior when induced with arabinose. This stochastic behavior is caused by the araE and araFGH genes encoding the arabinose transporters, because induction of the transporter genes by arabinose leads to a higher arabinose uptake and thus positive feedback (Siegele and Hu, 1997; Megerle et al., 2008). In addition, a negative feedback caused by fermentation of intracellular arabinose through the AraBAD enzymes leads to a non-gradual induction (Siegele and Hu, 1997). To avoid the negative and positive feedback, the araC gene and the araBAD and araFGH operons were deleted. Further, the low affinity arabinose transporter araE was put under the control of constitutive promoter P_cp8, as described (Khlebnikov et al., 2001; Kogenaru and Tans, 2014). The genotype of the resulting reporter strain U69 is P_leuO mVenus ΔleuO Δ(yjjP-yjjQ-bglJ) φ(ΔaraEp P_cp8 araE) Δ(araH-F) Δ(araC-araBAD) Δ(lacI-lacZYA) (Table 1). Using this strain the expression level of P_leuO mVenus was measured by flow-cytometry to quantify the cellular fluorescence in the population. Further, to ensure steady state conditions, cultures were grown in nutrient-poor tryptone medium. In this medium cultures that were inoculated from fresh overnight cultures to OD₆₀₀ of 0.05 reached an OD₆₀₀ of about 0.7–1 after 5 h of growth.

First, activation of the P_leuO mVenus fusion by BglJ-RcsB was tested. To this end, the reporter strain U69 was transformed with low-copy plasmid pKETS26 carrying bglJ under control of the IPTG-inducible P_UV5 promoter (P_UV5 bglJ, pSC-ori), and with plasmid pKES302 carrying bglJ under control of the arabinose-inducible P_BAD promoter (P_BAD bglJ, p15A-ori), respectively (Figure 2). Expression of bglJ was either not induced or induced by gradually increasing inducer concentrations. The analysis of P_leuO mVenus expression by flow-cytometry revealed that gradual induction of P_BAD bglJ expression (plasmid pKES302) with 2 μM–50 μM arabinose resulted in full activation of P_leuO mVenus even at the very low arabinose concentration of 2 μM (Figures 2B,C). Induction of P_BAD bglJ with 100 μM arabinose or higher concentrations caused growth defects. However, induction of P_UV5 bglJ with IPTG concentration ranging from 10 μM to 100 μM led to a gradual increase in expression of P_leuO mVenus and this increase was uniform in the population (Figures 2B,D). The presence of the P_UV5 bglJ or the P_BAD bglJ plasmids per se did not cause a significant increase in expression of P_leuO mVenus (Figures 2B–D). Likewise, IPTG or arabinose induction of transformants of the empty vectors pBAD30 and pKETS24, respectively, had no effect (Figure 2B). Taken together these data confirm activation of leuO transcription by BglJ-RcsB, they suggest that low cellular levels of BglJ are sufficient for activation, and that the P_UV5 bglJ plasmid is suitable for gradual induction of bglJ, while the P_BAD bglJ plasmid is not suitable.

Second, autoregulation of P_leuO mVenus by LeuO was analyzed using the leuO providing plasmids P_UV5 leuO (pKETS25, pSC-ori) and P_tac leuO (pKEHB27, pSC-ori) which carry leuO under control of the IPTG-inducible P_UV5 and P_tac promoters, respectively. In addition, a P_BAD leuO plasmid (pKES303, p15A-ori) was used. The promoter P_UV5 (carrying the UV5 mutation in the—10 box and the lacL8 mutation in the CRP-binding site) is ~10 times weaker than the P_tac promoter (Lanzer and Bujard, 1988), while the tightly regulated P_BAD leuO plasmid presumably directs similar levels of LeuO as the P_tac leuO plasmid considering that the P_BAD promoter is approximately 3 fold weaker than P_tac and that the copy number of the P_BAD plasmid (pKES303, p15A-ori) is ~3-fold higher than the copy number of the pSC-derived P_tac plasmid (Guzman et al., 1995). Flow cytometry revealed a slight increase in P_leuO mVenus expression at low levels of induction of plasmidic leuO (Figure 3). The data seem in agreement with weak positive autoregulation that was reported previously (Fang and Wu, 1998; Chen et al., 2003), but are statistically not significant (student's t-test, P-value > 0.05).

Next we addressed antagonistic regulation of P_leuO mVenus by BglJ-RcsB and by LeuO. To this end, the P_leuO mVenus reporter strain U69 was transformed with the two sets of leuO and bglJ expressing plasmids. First we analyzed antagonistic regulation of leuO transcription using the plasmid set, in which bglJ is expressed under control of the P_BAD promoter (P_BAD bglJ, pKES302) and leuO is expressed under control of the P_tac promoter (P_tac leuO, pKEHB27). Induction of bglJ expression with 2 μM–50 μM arabinose caused full activation of P_leuO mVenus (Figure 4), irrespective of the arabinose concentration, as shown above (Figure 2). Simultaneous induction of leuO by IPTG strongly reduced BglJ-RcsB-mediated activation of P_leuO mVenus, but even full induction of plasmidic leuO expression with 200 μM IPTG did not completely abrogate BglJ-RcsB-mediated activation (Figure 4). These results indicate that the level of BglJ provided by the P_BAD bglJ plasmid is above a threshold up to which LeuO can fully inhibit BglJ-RcsB activation. Since the P_BAD bglJ plasmid does not allow gradual activation, this plasmid set does not seem suitable for gradual induction of both regulators.

Second, we analyzed antagonistic regulation of P_leuO mVenus using the reverse set of plasmids that includes P_UV5 bglJ (pKETS26) and P_BAD leuO (pKES303) (Figure 5). With this set of plasmids expression levels of BglJ are lower and gradual induction of bglJ by IPTG resulted in a gradual increase in activation of the P_leuO mVenus fusion by BglJ-RcsB (Figure 5, compare with data in Figure 2). Simultaneous gradual induction of plasmidic P_BAD leuO with arabinose and of P_UV5 bglJ with IPTG led to a uniform decrease of expression of P_leuO mVenus in the whole population as compared to level of activation by BglJ-RcsB alone (Figure 5). Induction of leuO with an arabinose concentration of 50 μM was sufficient to completely abrogate activation by BglJ-RcsB (bottom right panel, Figure 5B). A plot of the median values of the flow cytometry results visualizes the gradual effects (Figure 5A).

Taken together, the data confirm that LeuO counteracts activation of the leuO promoter by BglJ-RcsB. Further, the data show that antagonistic regulation of the leuO promoters by LeuO and by BglJ-RcsB depends on the relative concentration of BglJ and LeuO, and the data indicate that BglJ-RcsB-mediated activation of P_leuO mVenus is inhibited by LeuO only if BglJ levels are rather low. The experimental data shown in Figure 5 were used to describe P_leuO activity in dependence of the concentration of BglJ and LeuO by a thermodynamic model based on Michaelis-Menten kinetics. In this model it was assumed that BglJ and LeuO regulate P_leuO independently of each other. Fitting of the function to the experimental data was significant (P-value < 0.001) (function plotted in Figure 6).

Next we addressed the relevance of the presumptive double-positive feedback regulation of leuO and bglJ by including the native gene of one of these two players, while providing the other one by the expression plasmid. In particular, we analyzed whether presence of the native yjjQ-bglJ operon that is activated by LeuO results in enhanced P_leuO mVenus expression, when LeuO is provided in trans. Second, we tested whether the presence of native leuO might affect activation of P_leuO by BglJ-RcsB.

For determining whether activation of the H-NS repressed yjjQ-bglJ operon by LeuO may yield sufficient BglJ protein for activation of P_leuO we compared P_leuO mVenus expression in (yjjQ-bglJ)⁺ strain U95 with expression in the isogenic Δ(yjjQ-bglJ) strain U69 (Figure 3). The data revealed no difference between wild-type yjjQ-bglJ⁺ strain U95 and Δ(yjjQ-bglJ) strain U69 suggesting that activation of yjjQ-bglJ by LeuO is either too low to provide sufficient levels of BglJ for activation of P_leuO mVenus or that LeuO interferes with activation by BglJ-RcsB. Second, we analyzed whether the presence of native leuO may affect activation of the leuO promoter by BglJ-RcsB. For this analysis the leuO gene was retained at its native locus and the fluorescence reporter gene mVenus was inserted downstream of leuO (as a transcriptional fusion) resulting in allele P_leuO leuO::mVenus in strain U97. Transformants of this strain with bglJ carrying plasmid pKETS26 (P_UV5 bglJ, pSC-ori), were grown with IPTG concentrations ranging from 10 μM to 200 μM and P_leuO leuO::mVenus expression was determined by flow cytometry. Comparison of the data obtained of P_leuO leuO::mVenus with the data obtained for P_leuO mVenus (ΔleuO) revealed no significant difference (Figures 2B,D). These data indicate that induction of the native leuO gene by BglJ does not provide sufficient LeuO to antagonize BglJ-RcsB-mediated activation of leuO.

Furthermore, we analyzed whether LeuO inhibits BglJ-RcsB-mediated activation of leuO transcription indirectly by downregulating BglJ-RcsB activity rather than by inhibiting activation of the leuO P2 promoter by BglJ-RcsB. To this end, activation of another BglJ-RcsB-activated promoter, the molR promoter (Salscheider et al., 2014), was analyzed in absence and presence of LeuO. BglJ was provided by P_UV5 bglJ plasmid pKETS26, and LeuO was provided by P_BAD leuO plasmid pKES303. As control, transformants with the empty vectors were analyzed in parallel. Activity of the molR promoter was determined using a P_molR mVenus reporter fusion. The expression analyses demonstrate that LeuO neither does affect activation of P_molR by BglJ-RcsB nor does LeuO-mediated activation of the native yjjQ-bglJ operon present in strain U76 lead indirectly to activation of P_molR (Figure 7). We note that induction of the P_BAD leuO with 50 μM arabinose resulted in slower growth to OD₆₀₀ = 0.6 after 5 h as compared to OD₆₀₀ = 1 which may explain the 1.5-fold reduce in basal expression of P_molR mVENUS in transformants of P_BAD leuO plasmid pKES303 and control plasmid P_UV5 pKETS24 (Figure 7).

Bacterial cultures of E. coli K-12 were grown in LB (10 g/l Bacto Tryptone, 5 g/l Bacto Yeast Extract, 5 g/l NaCl) or tryptone (10 g/l Bacto Tryptone, 5 g/l NaCl) media. Antibiotics were added with concentrations of 50 μg/ml ampicillin, 15 μg/ml chloramphenicol, and 25 μg/ml kanamycin. Strains, listed in Table 1, were constructed by transduction using phage T4GT7, by Red-Gam mediated gene deletion or gene replacement, and by homologous recombination, as described (Wilson et al., 1979; Hamilton et al., 1989; Datsenko and Wanner, 2000). Plasmids and their construction are listed in Table 2 and oligonucleotides are listed in Table 3. Standard molecular techniques, such as cloning, PCR, culture growth and induction of plasmid-provided genes, were performed according to standard protocols (Ausubel et al., 2005).

For expression analyses by flow cytometry cultures of transformants were inoculated from fresh overnight cultures to an OD₆₀₀ of 0.05 and grown for 5 h at 37°C in 10 ml tryptone medium containing antibiotics for selection of the plasmids. The cultures were diluted to OD₆₀₀ of 0.1 and kept on ice prior to analysis by flow cytometry. Flow cytometry was performed on a BD FACScalibur flow cytometer using CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA). For each sample, 50,000 events were measured at a rate between 500 and 1000 events per second. The experiments were repeated at least twice and representative sets of data are shown.

Fluorescence directed by the P_molR mVenus fusion was determined by Fluorescence spectroscopy using a CLARIOstar plate reader (BMG LABTECH, Germany). Briefly, cultures were grown as for flow cytometry and the fluorescence of cells equivalent to 1.5 OD₆₀₀ was measured using yellow fluorescent proteins specific excitation (495–515 nm) and detection (540–620 nm) channels. The average obtained of three biological replicates was calculated and the standard deviation is less than 25%.

To describe the transcription rate directed by PleuO in dependence of the concentration of BglJ and LeuO, a thermodynamic model based on Michaelis-Menten kinetics was used. In this model it was assumed that BglJ and LeuO regulate PleuO independently of each other. The binding probabilities were defined as B/(Bo+B) and L/(Lo+L), where B represents the concentration of BglJ in the cell, B0 the BglJ concentration at which the promoter is half occupied, L represents the concentration of LeuO and L0 the LeuO concentration at which the promoter is half occupied. Since LeuO acts as a repressor and BglJ as an activator of the leuO promoter four different states with a different expression rate were described. The basal expression level directed by PleuO in absence of BglJ and LeuO was defined as η0. In presence of LeuO and absence of BglJ, expression remains at a basal level defined as η0. However, in presence of BglJ but absence of LeuO, the expression level is higher which is defined as η1. When BglJ and LeuO are bound at the same time, the expression rate is defined as η0, because high levels of LeuO inhibit activation by BglJ, when BglJ is provided by the low-copy P_UV5 bglJ plasmid. Taking these four different states into account the expression rate of leuO in dependence of LeuO and BglJ concentration was described as

The function was fitted to the median expression values determined by flow cytometry (P_UV5 bglJ, and P_BAD leuO, Figure 5) using non-linear regression according to (Fox and Weisberg, 2011), which yielded a high fitting significance (P-value < 0.001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.