Extracytoplasmic Function σ Factors Can Be Implemented as Robust Heterologous Genetic Switches in Bacillus subtilis

Abstract

In bacteria, the promoter specificity of RNA polymerase is determined by interchangeable σ subunits. Extracytoplasmic function σ factors (ECFs) form the largest and most diverse family of alternative σ factors, and their suitability for constructing genetic switches and circuits was already demonstrated. However, a systematic study on how genetically determined perturbations affect the behavior of these switches is still lacking, which impairs our ability to predict their behavior in complex circuitry. Here, we implemented four ECF switches in Bacillus subtilis and comprehensively characterized their robustness toward genetic perturbations, including changes in copy number, protein stability, or antisense transcription. All switches show characteristic dose-response behavior that varies depending on the individual ECF-promoter pair. Most perturbations had performance costs. Although some general design rules could be derived, a detailed characterization of each ECF switch before implementation is recommended to understand and thereby accommodate its individual behavior.

Keywords: Biological Sciences; Biotechnology; Microbiology; Molecular Biology.

Graphical abstract

Figure 1

Choice of ECF Switches from Different Origins for Implementation in B. Subtilis (A) The ECF profiles of B. subtilis 168 (Bsu), B. licheniformis ATCC 14580 (Bli), B. cereus ATCC 10987 (Bce), E. coli K-12 DH10β (Eco), S. meliloti 1021 (Sme), and S. venezuelae ATCC 10712 (Sve) are represented. Boxes indicate that ECFs of the group indicated on top are present in the strain indicated on the left. The boxes are colored in black when ECF of that group and organism were tested in B. subtilis 168 and in gray when not tested. The ones shown in (C) are colored accordingly. Forty six ECFs were tested: B. licheniformis ATCC 14580 ECF41_BL00106 and ECFUC_BL04030; B. cereus environmental isolate ECF105_ecf105; E. coli K-12 DH10β ECF02_ECDH10B_2741 and ECF05_ ECDH10B_4491; S. meliloti 1021 ECF15_SMb21484 ECF16_SM_b20531, ECF26_SMa0143, ECF26_SMc02713, ECF26_SMc04051, ECF29_ SMb20592, ECF41_SM_b20030, and ECF42_SMc01150; and S. venezuelae ATCC 10712 ECF02_SVEN_4513, ECF12_SVEN_4870, ECF14_SVEN_4793, ECF17_SVEN_0063, ECF19_SVEN_0399, ECF20_SVEN_6501, ECF27_SVEN_3669, ECF38_SVEN_2914, ECF38_SVEN_3369, ECF38_SVEN_6611, ECF39_SVEN_3215, ECF39_SVEN_3278, ECF39_SVEN_3293, ECF39_SVEN_3759, ECF39_SVEN_4575, ECF41_SVEN_0136, ECF41_SVEN_0858, ECF41_SVEN_3295, ECF41_SVEN_3475, ECF41_SVEN_3480, ECF41_SVEN_3821, ECF41_SVEN_3859, ECF41_SVEN_1176, ECF42_SVEN_4377, ECF42_SVEN_7131, ECF50_SVEN_0980, ECF51_SVEN_0015, ECF52_SVEN_3871, ECF53_SVEN_0434, ECF53_SVEN_6745, ECF56_SVEN_4562, ECF121_SVEN_3185, and ECF123_SVEN_4540. (B) Generic genetic layout of the ECF switch. Thick arrows represent open reading frames. “T” represents terminators. Half circles represent ribosome-binding sites. Thin arrows represent promoters. (C) Dose-response curves drawn using the luminescence output value, represented through relative luminescence units (RLU) normalized by the optical density measured at 600 nm (OD600 nm), achieved 90 min after the addition of the inducer to the exponentially growing culture. ECF switches were built using ECFs BL00106 (red), BL04030 (green), ECF105 (blue), and SVEN_0399 (purple). Final concentrations of xylose used for induction of P_xylA were 0, 0.002, 0.008, 0.03, 0.125, or 0.5% (w/v). Vertical bars represent standard deviations calculated from three independent experiments.

Figure 2

Robustness of Heterologous ECF Switches in B. subtilis The four three-dimensional scatterplots show the behavior of the ECF switches upon the imposed alterations. Each plot corresponds to one ECF: BL00106 and BL04030 of B. licheniformis, ECF105 of B. cereus, and SVEN_0399 of S. venezuelae. The x axis corresponds to the baseline value of the switch; the y axis corresponds to the maximum output level of the switch, and the z axis corresponds to the maximal fold-induction. Baseline refers to the output observed in the absence of inducer, maximal output refers to the output value upon induction with the maximal concentration of inducer, and fold-induction refers to the ratio between maximal output and baseline values. Baseline and maximal output level are shown as relative luminescence units (RLU) normalized by the optical density at 600 nm (OD600 nm). All data points represent averages of three independent experiments in which cells were grown to exponential phase, the expression of the ECF was induced by 0.5% xylose (or 10 μg/mL of bacitracin for P_liaI-driven switches), and the values used were obtained 90 min after induction. The legend at the bottom of the figure shows the correspondence between the symbols and the performed alterations.