Fig. 1: Impact of neighboring transcription on HilD-mediated regulation.

Strains MA14341 (strain A), MA14694 (strain B), MA14692 (strain C) and MA14403 (strain D) carry a tetR-P^tet cassette (strain A) or a tetR-P^tettetA cassette (strains B, C, D) replacing the left portion of SPI-1 up to a position 1.2 Kb from hilD (∆[sitA-prgH1.2]::tetRA) and a translational hilA-GFP^SF fusion replacing most of SPI-1 material to the right of hilA (∆[hilA-STM2906]::GFP^SF; diagram in Supplementary Fig. 1). In strain C, the intrinsic transcription terminator from the histidine operon attenuator region (hisL) is inserted immediately downstream of tetA. In strain D, the translation initiation codon of tetA is changed to AAA. Only relevant promoters are shown (bent arrows). a−d Representative flow cytometry profiles of strains A, B, C and D, respectively, grown in the absence or in the presence of 0.4 µg/ml of AHTc. e Percentage of cells expressing hilA-GFP^SF in n = 4 independent repeats of the flow cytometry analysis. The error bars represent mean values ± SD. Statistical significance was determined by one-way ANOVA with Šidák’s multiple comparisons test. Adjusted P values for the untreated/+AHTc comparison were 0.9996, 0.0004, <0.0001 and 0.2582 in strains A, B, C and D, respectively. f Quantification of hilD mRNA from cultures of strains B, C and D grown in the presence or absence of AHTc. g Quantification of hilD mRNA from cultures of strain MA14888 grown in LB (untreated) or in LB supplemented with either AHTc, ARA or AHTc and ARA combined. Strain MA14888 carries the tetA initiator codon mutation in combination with the hisL terminator in the background of a nusG gene fusion to a promoter under the control of an ARA-inducible phage repressor (see text). In (f, g) RNA from n = 4 or n = 5 independent experiments was quantified by two-step reverse transcription-quantitative PCR (RT-qPCR). The RT step was carried out with a mixture of two gene-specific primers, AI69 annealing to hilD mRNA and AJ33 annealing to ompA mRNA. The resulting cDNA was amplified by qPCR with primers AI62-AI63 (hilD) and AJ32-AJ37 (ompA). Ct values were normalized to the Ct values determined for ompA. Error bars represent mean ± SD. Statistical significance was determined by one-way ANOVA with Šidák’s multiple comparisons test. Adjusted P values were 0.8268 and <0.0001 for the B/C and C/D comparisons, respectively, in (f), and 0.028 and 0.0006 for the untreated/+AHTc and +ARA/ + ARA+AHTc comparisons, respectively, in (g) (ns, P > 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). Source data are provided as a Source Data file.