Two Distinct Regulatory Systems Control Pulcherrimin Biosynthesis in Bacillus subtilis

Abstract

Regulation of transcription is a fundamental process that allows bacteria to respond to external stimuli with appropriate timing and magnitude of response. In the soil bacterium Bacillus subtilis, transcriptional regulation is at the core of developmental processes needed for cell survival. Gene expression in cells transitioning from exponential phase to stationary phase is under the control of a group of transcription factors called transition state regulators (TSRs). TSRs influence numerous developmental processes including the decision between biofilm formation and motility, genetic competence, and sporulation, but the extent to which TSRs influence bacterial physiology remains to be fully elucidated. Here, we demonstrate two TSRs, ScoC and AbrB, along with the MerR-family transcription factor PchR negatively regulate production of the iron chelator pulcherrimin in B. subtilis. Genetic analysis of the relationship between the three transcription factors indicate that all are necessary to limit pulcherrimin production during exponential phase and influence the rate and total amount of pulcherrimin produced. Similarly, expression of the pulcherrimin biosynthesis gene yvmC was found to be under control of ScoC, AbrB, and PchR and correlated with the amount of pulcherrimin produced by each background. Lastly, our in vitro data indicate a weak direct role for ScoC in controlling pulcherrimin production along with AbrB and PchR. The layered regulation by two distinct regulatory systems underscores the important, and somewhat enigmatic, role for pulcherrimin in B. subtilis physiology.

Keywords: Bacillus subtilis; pulcherrimin; transcription factor; transition state.

Fig. 1:. Pulcherrimin production in liquid (top) and solid (bottom) TSS media.

WT (DK1042) and isogenic mutants were grown in liquid TSS media or spotted (10 μL) onto solid TSS media and grown overnight at 37°C. The black scale marker corresponds to 5 mm.

Fig. 2:. ScoC, ArbB, and PchR Control Timing and Rate of Pulcherrimin Production.

A) Pulcherrimin production was measured as a function of growth phase, where T0 marks the transition from the exponential growth to stationary phase. Each panel represents the average A₄₁₀ for a given strain compared to the A₄₁₀ from WT (white squares). Error bars represent +/− the standard deviation. Lines running through the points are modeled using the drm function from the drc package in R (see Methods and Materials). B) Pulcherrimin production parameters as a function of genetic background: i) start of pulcherrimin production time relative to the transition phase of growth (T0), ii) the duration of pulcherrimin production, iii) the maximum estimated production rate, and iv) the maximum absorbance at 410 nm. For panels i–iii, brackets and asterisks indicate significant comparisons. For panel iv, brackets and “ns” indicate non-significant comparisons, where every other comparison had an adjusted p-value less than 0.05 as determined by T-test corrected for multiple comparisons with the Bonferroni correction.

Fig. 3:. ScoC, PchR, and AbrB Repress the yvmC Promoter.

A) Histograms representing fluorescence distribution as a function of cell count. In each panel, the negative control (grey, WT without GFP) was plotted with the corresponding genotype harboring a yvmC promoter fusion to GFP. Mean percent GFP positive with standard deviation in parentheses is provided to the right of each plot. Fluorescence was measured independently for each strain on three separate days with a representative shown. B) Median fluorescence for three separate trials (bar) with each trial median shown (circles). Error bars represent stand deviation between trials. Asterisks indicate an adjusted p-value less 0.05 while “ns” indicate non-significant comparisons with WT.

Fig. 4:. DNAse I Protection Varies Among ScoC, PchR, and AbrB.

Electropherograms of fluorescent DNAse I footprinting analysis of PchR (A), AbrB (B), and ScoC (C) with fluorescently labeled yvmC promoter as a function of estimated nucleotide position. Fluorescence intensity (RFU, panel i) of reactions incubated with (blue) and without protein (red). Differential peak height (panel ii) between reactions with protein and without protein. Differential peak heights less than zero indicate protection while differential peak heights greater than zero hypersensitivity.

Fig. 5:. ScoC, PchR, and AbrB Bind Near the Core Promoter Region of yvmC.

Electrophoretic mobility shift assays with WT P_yvmC (A) and the Δ59 promoter P_yvmCΔ59 (B) with increasing concentrations of purified PchR (left panel), AbrB (middle panel), and ScoC (right panel). Unshifted bands are marked with unfilled triangles, shifted bands are marked with filled triangles, and smears are marked with brackets. Proteins were diluted two-fold and final concentrations are as follows: PchR (15.6 to 250 nM for WT, 125–250 nM for Δ59), AbrB (62.5 to 1000 nM for WT, 500–1000 nM for Δ59), and ScoC (125 to 1000 nM for WT, 250–1000 nM for Δ59).

Fig. 6:. Model of Pulcherrimin Regulation by ScoC, AbrB, and PchR.

A) Pulcherriminic acid biosynthesis by the cyclization of tRNA-charged leucines to form cyclo(L-leucine-leucine) and the subsequent oxidation by CypX to form water-soluble pulcherriminic acid. Pulcherriminic acid is then transported out of the cell by YvmA, where it can form the insoluble pulcherrimin complex with iron, which forms a red color and has a peak absorbance at 410 nm. B) During exponential growth, PchR, AbrB, and ScoC bind directly to the yvmC promoter to inhibit expression of yvmC-cypX. Our data suggests ScoC can bind to the promoter but does so weakly. Thus, it is possible its mode of regulation may be direct or indirect through an unidentified transcriptional regulator controlled by ScoC. In any case, as nutrients become limiting, the transition state regulators become inactive, relieving repression on the yvmC promoter. As a MarR family transcription factor, PchR activity is likely regulated by a small ligand of which the identity is currently not known.